Recombinant modified vaccinia ankara (MVA) vaccinia virus containing restructured insertion sites

ABSTRACT

The present invention relates to recombinant modified vaccinia Ankara (MVA) virus containing restructured sites useful for the integration of heterologous nucleic acid sequences into an intergenic region (IGR) of the virus genome, where the IGR is located between two adjacent, essential open reading frames (ORFs) of the vaccinia virus genome, wherein the adjacent essential ORFs are non-adjacent in a parental MVA virus used to construct the recombinant MVA virus, and to related nucleic acid constructs useful for inserting heterologous DNA into the genome of a vaccinia virus, and further to the use of the disclosed viruses as a medicine or vaccine.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage application under 35 U.S.C. 371 ofPCT Application No. PCT/US2010/052929 having an international filingdate of 15 Oct. 2010, which designated the United States, which PCTapplication claimed the benefit of U.S. Provisional Application No.61/252,326 filed 16 Oct. 2009, the entire disclosure of each of which isincorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronictext file named “Seq_Listing_(—)6137NIAID-22-PCT_ST25.txt”, having asize in bytes of 42 KB, and created on Oct. 15, 2010. The informationcontained in this electronic file is hereby incorporated by reference inits entirety pursuant to 37 CFR §1.52(e)(5).

FIELD OF THE INVENTION

The present invention relates to insertion sites useful for the stableintegration of heterologous DNA sequences into the MVA genome. Morespecifically, the invention relates to methods of restructuring regionsof the modified vaccinia Ankara (MVA) virus genome that contain acombination of essential and non-essential gene, so that heterologousDNA remains stably integrated into the genome.

BACKGROUND

The members of the poxvirus family have large double-stranded DNAgenomes encoding several hundred proteins (Moss, B. 2007 “Poxyiridae:The Viruses and Their Replication” in Fields Virology, 5^(th) Ed. (D. M.Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B.Roizman, and S. E. Straus, Eds), Lippincott Williams & Wilkins,Philadelphia, Pa.). Poxviruses are divided into the subfamiliesChordopoxyirinae and Entomopoxyirinae, based on vertebrate and insecthost range. The subfamily Chordopoxyirinae consists of eight genera:Orthopoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus,Suipoxvirus, Molluscipoxvirus, and Yatapoxvirus. The prototypal memberof the genus Orthopoxvirus is vaccinia virus. Vaccinia virus (VACV), thefirst recombinant virus shown to induce a protective immune responseagainst an unrelated pathogen (Moss, B., G. L. Smith, J. L. Geria, andR. H. Purcell. 1984. Live recombinant vaccinia virus protectschimpanzees against hepatitis B. Nature 311:67-69; Paoletti, E., B. R,Lipinskas, C. Samsonolf, S. R. Mercer, and D. Panicali. 1984.Construction of live vaccines using genetically engineered poxviruses;biological activity of vaccinia virus recombinants expressing thehepatitis B virus surface antigen and the herpes simplex virusglycoprotein D. Proc. Natl. Acad. Sci. USA 81:193-197), is beingemployed as a vector for veterinary and wildlife vaccines (Moss, B.1996. Genetically engineered poxviruses for recombinant gene expression.vaccination, and safety. Proc. Natl. Acad. Sci. USA 93:11341-11348).Development of recombinant VACV for human use, however, has been impededby safety concerns. For this reason, there is interest in modified VACVAnkara (MVA), a highly attenuated smallpox vaccine with an exemplarysafety profile even in immunodeficient animals (Mayr, A., V.Hochstein-Mintzel, and H. Stickl. 1975. Passage history, properties, andapplicability of the attenuated vaccinia virus strain MVA. Infection3:6-14. (In German); Stickl, H., V. Hochstein-Mintzel, A. Mayr, H. C.Huber, H. Schafer, and A. Holzner. 1974. MVA vaccination againstsmallpox: clinical trial of an attenuated live vaccinia virus strain(MVA). Dtsch. Med. Wschr. 99:2386-2392 (In German); Stittelaar, K. J.,T. Kuiken, R. L. de Swart, G. van Amerongen, H. W. Vos, H. G. Niesters,P. van Schalkwijk, T. van der Kwast, L. S. Wyatt, B. Moss, and A. D.Osterhaus. 2001. Safety of modified vaccinia virus Ankara (MVA) inimmune-suppressed macaques. Vaccine 19:3700-3709). The genomic sequenceof MVA (Mayr, A. et al. 1978 Zentralbl Bakteriol 167:375-390), whichcannot grow in most mammalian cells and which is a good candidate for arecombinant vaccine vector, is known (Sutter, G. and Moss, B. 1992 ProcNatl Acad Sci USA 89:10847-10851; and Sutter, G. et al. 1994 Vaccine12:1032-1040) has been passaged over 570 times in chicken embryofibroblasts, during which six major deletions relative to the parentalwild-type strain Ankara, accompanied by a severe restriction in hostrange, have occurred (Meyer, H. et al. 1991 J Gen Virol 72:1031-1038).MVA is severely host range restricted and propagates poorly or not atall in most mammalian cells because of a block in virion assembly(Sutter, G., and B. Moss. 1992. Nonreplicating vaccinia vectorefficiently expresses recombinant genes. Proc. Natl. Acad. Sci. USA89:10847-10851). Initial experiments with recombinant MVA (rMVA)demonstrated its ability to robustly express foreign proteins (Sutter,G., and B. Moss. 1992. Nonreplicating vaccinia vector efficientlyexpresses recombinant genes. Proc. Natl. Acad. Sci. USA 89:10847-10851)and induce protective humoral and cell-mediated immunity (Sutter, G., L.S. Wyatt, P. L. Foley, J. R. Bennink, and B. Moss. 1994. A recombinantvector derived from the host range-restricted and highly attenuated MVAstrain of vaccinia virus stimulates protective immunity in mice toinfluenza virus. Vaccine 12:1032-1040). Currently, rMVA candidatevaccines expressing genes from a wide variety of pathogens areundergoing animal and human testing (Gomez, C. E., J. L. Najera, M.Krupa, and M. Esteban. 2008. The poxvirus vectors MVA and NYVAC a genedelivery systems for vaccination against infection diseases and cancer.Curr. Gene Ther. 8:97-120).

While developing candidate human immunodeficiency virus (HIV) and othervaccines, it was observed that mutant rMVA loses the ability to expressforeign proteins after tissue culture passage (Stittelaar, K. J., L. S.Wyatt, R. L de Swart, H. W. Vos, J. Groen, G. van Amerongen, R. S. vanBinnendijk, S. Rozenblatt, B. Moss. and A. Osterhaus. 2000. Protectiveimmunity in macaques vaccinated with a modified vaccinia virusAnkara-based measles virus vaccine in the presence of passively acquiredantibodies. J. Virol, 74:4236-4243; Wyatt, L. S., I. M. Belyakov, P. L.Earl, J. A. Berzofsky, and B. Moss. 2008. Enhanced cell surfaceexpression, immunogenicity and genetic stability resulting from aspontaneous truncation of HIV Env expressed by a recombinant MVA.Virology 372:260-272; Wyatt, L. S., S. T. Shors, B. R. Murphy, and B.Moss. 1996. Development of a replication-deficient recombinant vacciniavirus vaccine effective against parainfluenza virus 3 infection in ananimal model. Vaccine 14:1451-1458). This instability may initially goundetected, however, unless individual plaques are isolated andanalyzed. Nevertheless, once established in the population, thenonexpressors can rapidly overgrow the original rMVA. Theseconsiderations are particularly important for production of largevaccine seed stocks of rMVA. The instability of cloned genes in MVA issurprising, since MVA had already undergone genetic changes during itsadaptation through hundreds of passages in chicken embryo fibroblasts(CEFs) and is now quite stable. Indeed, identical 167,000-bp genomesequences have been reported for three independent plaque isolates,accession numbers U94848, AY603355, and DQ983236, and by Antoine et al.(Antoine, G., F. Scheiflinger, F. Dorner, and F. G. Falkner. 2006.Corrigendum 10 “The complete genomic sequence of the modified vacciniaAnkara (MVA) strain: comparison with other orthopoxviruses.” Virology350:501-502. [Correction to 244:365, 1998.]). Although the cause of theinstability of the gene inserts had not been previously investigated,harmful effects of the recombinant protein seem to play a role in theselective advantage of nonexpressing mutants. Thus, reducing theexpression level of parainfluenza virus and measles virus transmembraneproteins and deleting part of the cytoplasmic tail of HIV Env improvesthe stability of rMVAs (Stittelaar, K. J., L. S. Wyatt, R. L. de Swart,H. W. Vos, J. Groen, G. van Amerongen, R. S. van Binnendijk, S.Rozenblatt, B. Moss. and A. Osterhaus. 2000. Protective immunity inmacaques vaccinated with a modified vaccinia virus Ankara-based measlesvirus vaccine in the presence of passively acquired antibodies. J.Virol, 74:4236-4243; Wyatt, L. S., I. M. Belyakov, P. L. Earl, J. A.Berzofsky, and B. Moss. 2008. Enhanced cell surface expression,immunogenicity and genetic stability resulting from a spontaneoustruncation of HIV Env expressed by a recombinant MVA. Virology372:260-272; Wyatt, L. S., S. T. Shors, B. R. Murphy, and B. Moss. 1996.Development of a replication-deficient recombinant vaccinia virusvaccine effective against parainfluenza virus 3 infection in an animalmodel. Vaccine 14:1451-1458). Reducing expression, however, can alsodecrease immunogenicity and therefore may be undesirable (Wyatt, L. S.,P. L. Earl, J. Vogt, L. A. Eller, D. Chandran, J. Liu, H. L. Robinson,and B. Moss. 2008. Correlation of immunogenicities and in vitroexpression levels of recombinant modified vaccinia virus Ankara HIVvaccines. Vaccine 26:486-493).

In view of the potential value of rMVA as a vaccine, it is important tounderstand this pernicious instability problem, and to develop methodsfor constructing stable, recombinant MVA viruses. Additionally, anunderstanding of the stability problem might provide insights that haveapplication to other DNA expression vectors. The present inventionprovides such insights and provides for a solution to the problem ofconstructing stable, recombinant MVA viruses.

SUMMARY OF THE INVENTION

The present invention relates to the discovery that the genome of amodified vaccinia Ankara (MVA) virus can be made more stable byrestructuring regions of the genome. In particular, the inventors havediscovered that regions of the genome containing non-essential genes aregenetically unstable. Moreover such regions can be made more stable byremoving non-essential DNA, and making essential genes in these regionsadjacent to one another. Because loss of essential genes results in avirus having a growth disadvantage, such viruses are quickly lost fromthe population resulting in a population of viruses in which theessential genes, and any intervening DNA, is maintained.

The disclosure provides a recombinant modified vaccinia Ankara (MVA)virus comprising a heterologous nucleic acid sequence located betweentwo adjacent, essential open reading frames of the MVA virus genome. Thechoice of essential ORFs is such that the ORFs are non-adjacent in thegenome of a parental MVA virus used to construct the recombinant virusesof the present invention. That is, the essential ORFs are separated byat least one non-essential ORF. However, in the recombinant modifiedvaccinia Ankara (MVA) progeny virus, the essential ORFs have been madeadjacent. That is, there are no intervening, non-essential ORFs betweenthe essential ORFs. Consequently, the region between the essential ORFsis stable, and is maintained in the virus population. Consequently, thisregion provides a new and useful site for the insertion of heterologousnucleic acid sequences. Such heterologous nucleic acid sequences canencode therapeutically useful proteins, such as antigens.

The disclosure also provides nucleic acid constructs that can be used toconstruct recombinant modified vaccinia Ankara (MVA) viruses of thepresent invention. Such constructs contain essential ORFs from theparental MVA virus, and that are non-adjacent in the parental virus.However, in the disclosed nucleic acid constructs, these essential ORFshave been made adjacent to one another. Moreover, constructs aredisclosed that contain intergenic regions between the essential ORFs,which can be used for the insertion of heterologous nucleic acidsequences.

Finally, also disclosed are methods of using viruses of the presentinvention for the prevention and treatment of disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Phylogenetic relationships of HIV-1 and HIV-2 based on identityof pol gene sequences. SIV_(cpz) and SIV_(smm) are subhuman primatelentiviruses recovered from a chimpanzee and sooty mangabey monkey,respectively.

FIG. 2. Phylogenetic relationships of HIV-1 groups M, N and O with fourdifferent SIV_(cpz) isolates based on full-length pol gene sequences.The bar indicates a genetic distance of 0.1 (10% nucleotide divergence)and the asterisk positions group N HIV-1 isolates based on envsequences.

FIG. 3. Tropic and biologic properties of HIV-1 isolates.

FIG. 4. HIV-encoded proteins. The location of the HIV genes, the sizesof primary translation products (in some cases polyproteins), and theprocessed mature viral proteins are indicated.

FIG. 5. Schematic representation of a mature HIV-1 virion.

FIG. 6. Linear representation of the HIV-1 Env glycoprotein. The arrowindicates the site of gp160 cleavage to gp120 and gp41. In gp120,cross-hatched areas represent variable domains (V₁ to V₅) and open boxesdepict conserved sequences (C₁ to C₅). In the gp41 ectodomain, severaldomains are indicated: the N-terminal fusion peptide, and the twoectodomain helices (N- and C-helix). The membrane-spanning domain isrepresented by a black box. In the gp41 cytoplasmic domain, theTyr-X-X-Leu (YXXL) endocytosis motif (SEQ ID NO: 1) and two predictedhelical domains (helix-1 and -2) are shown. Amino acid numbers areindicated.

FIG. 7. pLW-73 nucleic acid construct (SEQ ID NO:2 and 3).

FIG. 8. Nucleotide sequence of the pLW-73 transfer vector (top strand,SEQ ID NO: 2; bottom strand, SEQ ID NO: 3).

FIG. 9. Nucleotide sequence encoding Ugandan Glade D Env protein(isolate AO7412) (SEQ ID NO: 4).

FIG. 10. Codon altered nucleotide sequence encoding Ugandan clade Dgagpol protein (isolate AO3349) (SEQ ID NO: 5).

FIG. 11. Generation of recombinant MVAs and analysis of stability ofinserted genes. A) Schematic diagram of insertion of env and gagpol intoDel II and Del III sites, respectively. B) Evaluation of stability byimmunostaining.

FIG. 12. Types and frequency of env mutations in MVA/65A/G env.

FIG. 13. Insertion of Env in I8R/G1L IGR and Gag Pol in Del III.

FIG. 14. Modifications to A/G constructs to increase stability.

FIG. 15. Env expression after plaque passages.

FIG. 16. PCR and Western blot analysis of individual clones.

FIG. 17. Expression of A/G env by double recombinant MVA.

FIG. 18. Recombinant viruses expressing env and gagpol from UgandanHIV-1 isolates.

FIG. 19. MVA/UGD4a—analysis of non-staining env plaques.

FIG. 20. Modification of UGD env gene in recombinant MVA.

FIG. 21. MVA/UGD4b—analysis of non-staining gag plaques. *, location ofruns of 4-6 G or C residues.

FIG. 22. Modification of UGD gagpol gene in recombinant MVA.

FIG. 23. Construction of stable recombinant MVA expressing UGD env andgagpol.

FIG. 24. Cellular responses elicited by MVA/UGD4d.

FIG. 25. Antibody responses elicited by MVA/UGD4d.

FIG. 26. Outline of method for restructuring the del III site of MVAvirus genome.

FIG. 27 pLW-76 nucleic acid construct (SEQ ID NO:21 and 22).

FIG. 28. Syncytial phenotype in rMVA due to restructuring of the del IIIsite

FIG. 29 Comparison of heterologous nucleic acid stability in differentrecombinant MVA viruses

FIG. 30 Comparison of UGD Env protein expression level in differentrecombinant MVA viruses

FIG. 31 Nucleotide sequence of the pLW-76 transfer vector (SEQ ID NO:21and 22).

DEPOSIT OF MICROORGANISM

The following microorganism has been deposited in accordance with theterms of the Budapest Treaty with the American Type Culture Collection(ATCC), Manassas, Va., on the date indicated:

Microorganism Accession No. Date MVA 1974/NIH Clone 1 PTA-5095 Mar. 27,2003

MVA 1974/NIH Clone 1 was deposited as ATCC Accession No.: PTA-5095 onMar. 27, 2003 with the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110-2209, USA. This deposit was madeunder the provisions of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure and the Regulations thereunder (Budapest Treaty). This assuresmaintenance of a viable culture of the deposit for 30 years from date ofdeposit. The deposit will be made available by ATCC under the terms ofthe Budapest Treaty, and subject to an agreement between Applicant andATCC which assures permanent and unrestricted availability of theprogeny of the culture of the deposit to the public upon issuance of thepertinent U.S. patent or upon laying open to the public of any U.S. orforeign patent application, whichever comes first, and assuresavailability of the progeny to one determined by the U.S. Commissionerof Patents and Trademarks to be entitled thereto according to 35 USC§122 and the Commissioner's rules pursuant thereto (including 37 CFR§1.14). Availability of the deposited strain is not to be construed as alicense to practice the invention in contravention of the rights grantedunder the authority of any government in accordance with its patentlaws.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the claims.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. See, e.g., Singleton P andSainsbury D., Dictionary of Microbiology and Molecular Biology, 3rd ed.,J. Wiley & Sons, Chichester, N.Y., 2001 and Fields Virology, 5^(th) Ed.(D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B.Roizman, and S. E. Straus, eds), Lippincott Williams & Wilkins,Philadelphia, Pa., 2007. Although any methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the present invention, the preferred methods and materialsare now described. All publications mentioned herein are incorporatedherein by reference to disclose and describe the methods and/ormaterials in connection with which the publications are cited.

According to the present invention, an isolated protein, or nucleic acidmolecule, is a protein, or nucleic acid molecule, that has been removedfrom its natural milieu. An isolated protein, or nucleic acid molecule,can, for example, be obtained from its natural source, be produced usingrecombinant DNA technology, or be synthesized chemically. As such,isolated does not reflect the state or degree to which a protein ornucleic acid molecule is purified.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It should be understood that as used herein, the term “a” entity or “an”entity refers to one or more of that entity. For example, a nucleic acidmolecule refers to one or more nucleic acid molecules. As such, theterms “a”, “an”, “one or more” and “at least one” can be usedinterchangeably. Similarly the terms “comprising”, “including” and“having” can be used interchangeably.

The transitional term “comprising” is synonymous with “including,”“containing,” or “characterized by,” is inclusive or open-ended and doesnot exclude additional, unrecited elements or method steps.

The transitional phrase “consisting of” excludes any element, step, oringredient not specified in the claim, but does not exclude additionalcomponents or steps that are unrelated to the invention such asimpurities ordinarily associated therewith.

The transitional phrase “consisting essentially of” limits the scope ofa claim to the specified materials or steps and those that do notmaterially affect the basic and novel characteristic(s) of the claimedinvention.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates, which may need to be independently confirmed.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodiments arespecifically embraced by the present invention and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed. In addition, all sub-combinations are also specificallyembraced by the present invention and are disclosed herein just as ifeach and every such sub-combination was individually and explicitlydisclosed herein.

Complete genome sequences have been reported for at least one member ofeach chordopoxvirus genus and two entomopoxviruses. Nearly 100 genes areconserved in all chordopoxviruses, and about half of these are alsopresent in entomopoxviruses. Based on the above, several generalizationscan be made: Genes are largely nonoverlapping, tend to occur in blockspointing toward the nearer end of the genome, are usually located in thecentral region if highly conserved and concerned with essentialreplication functions, and are usually located in the end regions ifvariable and concerned with host interactions. The arrangement of thecentral genes is remarkably similar in all chordopoxviruses. Aconvention for naming vaccinia virus genes or ORFs (open readingframes), originating prior to sequencing the entire genome andsubsequently used for the complete sequence of the Copenhagen strain ofvaccinia virus, consists of using the HindIII restriction endonucleaseDNA fragment letter, followed by the ORF number (from left to right)within the fragment, and L or R, depending on the direction of the ORF.An exception to this rule was made for the HindIII C fragment; the ORFswere numbered from the right in order to avoid starting at the highlyvariable left end of the genome. Polypeptide names correspond to genenames, except that L or R is dropped. In most subsequent completepoxvirus genome sequences, ORFs were numbered successively from one endof the genome to the other. Nevertheless, the old letter designationshave been retained as common names to provide continuity in theliterature. The ORF number of the Western Reserve (WR) strain ofvaccinia virus is commonly shown in reference books because this strainhas been used for the great majority of biochemical and genetic studies.

The inventors of the present invention have identified new sites, andmethods for creating new sites, for the stable insertion of exogenousDNA sequences into the genome of modified vaccinia Ankara (MVA) virus.The present invention resulted from work aimed at identifying methods ofconstructing stable, recombinant MVA viruses. It had previously beenobserved that while recombinant MVAs containing heterologous DNAsequences inserted into the MVA genome could be obtained, theseinsertions were often unstable. Investigations of this instabilityyielded the conclusion that the insertion of heterologous DNA sequencesnon-essential for viral propagation into spaces between ORFs could beexpected to be deleted by the virus as well. Thus was recognized a needfor improved strategies for constructing stable, recombinant MVAviruses.

As used herein, an open reading frame (ORF) means a string of contiguousnucleotides that encode the amino acids of a protein. Such proteins canbe peptides, polypeptides, and can be any length greater than a singleamino acid. It should be understood that an ORF may also include a stopcodon, even though such codon does not encode an amino acid. It will beappreciated by those skilled in the art that, due to recombinationevents, some ORFs have lost portions of their original coding capacityand thus encode proteins that are non-functional. Such ORFs aresometimes referred to as ORF fragments. ORFs do not include regulatoryelements (e.g., promoters, transcriptional control elements, enhancers,etc.) that are located outside of the coding region. In contrast, a generefers to an ORF (including the stop codon) and regulatory elementscapable of regulating transcription of the ORF.

ORFs can be referred to as adjacent or non-adjacent. As used herein, twoORFs are adjacent when they reside in the same nucleic acid molecule,and their two closest ends are not separated by another poxvirus ORF.Non-adjacent ORFs are ORFs whose two closest ends are separated byanother poxvirus ORF. Adjacent ORFs can be contiguous, meaning thatthere is no other nucleotide sequence between a terminal codon belongingto one ORF and a terminal codon belonging to the other ORF. A terminalcodon means the first or last codon of an ORF, including the stop codon.One example of a terminal codon is the codon encoding the first 5′ aminoacid of the protein encoded by the ORF. Another example of a terminalcodon is the codon encoding the last 3′ amino acid of the proteinencoded by the ORF. Still another example of a terminal codon is thestop codon for the ORE.

Adjacent ORFs can also be separated by a nucleic acid sequence. Such asequence is referred to as an intergenic region. As used herein anintergenic region means a nucleic acid sequence between the closestterminal codons of adjacent ORFs that does not contain nucleotidesequences derived from vaccinia virus, other than poxvirustranscriptional control elements. IGR sequences lie outside the stopcodons of adjacent ORFs and thus do not encode any portion of theprotein encoded by the adjacent ORFs. IGR sequences may contain poxvirustranscriptional control elements. IGRs may also contain sequencesderived from organisms other than a poxvirus. Preferably IGRs are freeof any poxvirus sequences that are not part of a poxvirustranscriptional control element. In one embodiment, the IGR comprises atleast one heterologous nucleic acid sequence. Such sequence can beinserted at a restriction enzyme recognition site, or restriction site,which is naturally present in the IGR or which has been introduced intothe IGR for the purpose of inserting other heterologous nucleic acidsequences.

While the nucleotide sequences of ORFs encode proteins, the intergenicregions (IGRs) between two ORFs have no coding capacity. Thus they mayserve as sites into which heterologous DNA can be inserted withoutaffecting the production of any viral proteins. IGRs may, however,comprise regulatory elements, binding sites, promoter and/or enhancersequences essential for or involved in the transcriptional control ofthe viral gene expression. Thus, the IGR may be involved in theregulatory control of the viral life cycle. Even so, the inventors havefound that the IGR's can be used to stably insert heterologous nucleicacid sequences into the MVA genome without influencing or changing thetypical characteristics and gene expression of MVA. The new insertionsites are especially useful, since no ORF or coding sequence of MVA isaltered.

Before further describing the invention, it is useful to have anunderstanding of the arrangement of genes in the poxvirus genome. Thenucleotide sequence of an ORF regularly starts with a start codon andends with a stop codon. Depending on the orientation of the two adjacentORFs the IGR, the region in between these ORFs, is flanked either by thetwo stop codons of the two adjacent ORFs, or, by the two start codons ofthe two adjacent ORFs, or, by the stop codon of the first ORF and thestart codon of the second ORF, or, by the start codon of the first ORFand the stop codon of the second ORF.

Accordingly, the insertion site for the exogenous DNA sequence into theIGR may be downstream or 3′ of the stop codon of a first ORF. In casethe adjacent ORF, also termed second ORE, has the same orientation asthe first ORF, this insertion site downstream of the stop codon of thefirst ORF lies upstream or 5′ of the start codon of the second ORF.

In case the second ORF has an opposite orientation relative to the firstORF, which means the orientation of the two adjacent ORFs points to eachother, then the insertion site lies downstream of the stop codons ofboth ORFs.

As a third alternative, in case the two adjacent ORFs read in oppositedirections, but the orientation of the two adjacent ORFs points awayfrom each other, which is synonymous with a positioning that ischaracterized in that the start codons of the two ORFs are adjacent toeach other, then the exogenous DNA is inserted upstream relative to bothstart codons.

ORFs in the MVA genome occur in two coding directions. Consequently,mRNA synthesis activity occurs from left to right, i.e., forwarddirection and, correspondingly, from right to left (reverse direction).It is common practice in poxvirology and it became a standardclassification for vaccinia viruses to identify ORFs by theirorientation and their position on the different HindIII restrictiondigest fragments of the genome. For the nomenclature, the differentHindIII fragments are named by descending capital letters correspondingwith their descending size. The ORF are numbered from left to right oneach HindIII fragment and the orientation of the ORF is indicated by acapital L (standing for transcription from right to Left) or R (standingfor transcription from left to Right). Additionally, there is a morerecent publication of the MVA genome structure, which uses a differentnomenclature, simply numbering the ORF from the left to the right end ofthe genome and indicating their orientation with a capital L or R(Antoine, G. et al. 1998 Virology 244:365-396). As an example the I8RORF, according to the old nomenclature, corresponds to the 069R ORFaccording to Antoine et al.

In their efforts to make recombinants of modified vaccinia virus Ankara(MVA) expressing HIV genes as candidate vaccines, the inventorsdetermined that one of the causes of instability is due to deletions ofthe foreign gene and flanking MVA sequences. In an attempt to overcomethis problem they set out to insert foreign genes between conservedgenes in order to prevent viable deletions from occurring in recombinantMVAs. Viruses with such deletions have a growth advantage and will thusovergrow rMVA virus populations. If one inserts foreign genes betweenconserved genes in the vaccinia genome (these genes are considered to berequired for vaccinia virus replication and are therefore “essentialgenes”), any deletion of an essential gene would inhibit virusreplication, and, therefore, not overgrow the recombinant MVAs. Thus,the stable expression of the rMVA population is maintained. The strainof MVA that the inventors have been using to make their recombinants wasprovided by them to the Centers for Disease Control and Prevention (CDC)and was subsequently sequenced by Acambis (Genbank Accession numberAY603355). The strain of MVA that Bavarian Nordic has based theirWO03/097845 publication on is vaccinia virus strain modified vacciniaAnkara (Genbank Accession number U94848) sequenced by Antoine, G. et al.1998 Virology 244:365-396. (Note that the gene numbers in these twosequences for a given gene are different.)

The inventors initially looked at genes conserved in the Poxviridaefamily as well as those genes conserved in subfamily Chordopoxyirinae(the vertebrate poxviruses) (Upton, C. et al. 2003 Journal of Virology77:7590-7600). These genes are listed in the nomenclature of Copenhagenvaccinia virus (Genbank Accession number M35027) given on the PoxvirusBioinformatics Resource Center found on the world wide web atpoxvirus.org. These genes total 49 conserved genes in the Poxvirusfamily and 41 additional genes conserved in chordopoxviruses, making atotal of 90 conserved genes. From these 90 conserved genes, theinventors listed intergenic sites between conserved gene pairs. Thesegene pairs are listed below in Table 1. (Note that genes are marked thathave not been included in the Bavarian Nordic WO03/097845 publication).

TABLE 1 Intergenic Sites between Conserved Genes Listed in Genes/CDC/Acambis Antoine WO03/097845 publ ? Copenhagen Genes et al. Genes N =No F9L-F10L 040-041 038L-039L F12L-F13L 044-045 042L-043L N F17R-E1L049-050 047R-048L N E1L-E2L 050-051 048L-049L N E8R-E9L 057-058055R-056L E9L-E10R 058-059 056L-057L N I1L-I2L 064-065 062L-063L NI2L-I3L 065-066 063L-064L N I5L-I6L 068-069 066L-067L I6L-I7L 069-070067L-068L N I7L-I8R 070-071 068L-069R N I8R-G1L 071-072 069R-070L NG1L-G3L 072-073 070L-071L N G3L-G2R 073-074 071L-072R N G2R-G4L 074-075072R-073L N G4L-G5R 075-076 073L-074R N G5R-G5.5R 076-077 074R-075R NG5.5R-G6R 077-078 075R-076R N G6R-G7L 078-079 076R-077L N G7L-G8R079-080 077L-078R G8R-G9R 080-081 078R-079R G9R-L1R 081-082 079R-080R NL1R-L2R 082-083 080R-081R L2R-L3L 083-084 081R-082L L3L-L4R 084-085082L-083R L4R-L5R 085-086 083R-084R N L5R-J1R 086-087 084R-085R NJ3R-J4R 089-090 087R-088R N J4R-J5L 090-091 088R-089L J5L-J6R 091-092089L-090R J6R-H1L 092-093 090R-091L N H1L-H2R 093-094 091L-092R NH2R-H3L 094-095 092R-093L H3L-H4L 095-096 093L-094L N H4L-H5R 096-097094L-095R H5R-H6R 097-098 095R-096R N H6R-H7R 098-099 096R-097R H7R-D1R099-100 097R-098R D1R-D2L 100-101 098R-099L N D2L-D3R 101-102 099L-100RN D3R-D4R 102-103 100R-101R N D4R-D5R 103-104 101R-102R D5R-D6R 104-105102R-103R N D6R-D7R 105-106 103R-104R D9R-D10R 108-109 106R-107R ND10R-D11L 109-110 107R-108L D11L-D12L 110-111 108L-109L D12L-D13L111-112 109L-110L D13L-A1L 112-113 110L-111L A1L-A2L 113-114 111L-112L NA2L-A2.5L 114-115 112L-113L N A2.5L-A3L 115-116 113L-114L A3L-A4L116-117 114L-115L A4L-A5R 117-118 115L-116R A5R-A6L 118-119 116R-117L NA6L-A7L 119-120 117L-118L A7L-A8R 120-121 118L-119R A8R-A9L 121-122119R-120L N A9L-A10L 122-123 120L-121L N A10L-A11R 123-124 121L-122R NA11R-A12L 124-125 122R-123L A12L-A13L 125-126 123L-124L A13L-A14L126-127 124L-125L A14L-A14.5L 127-128 125L-125.5L N A14.5L-A15L 128-129125.5L-126L N A15L-A16L 129-130 126L-127L N A16L-A17L 130-131 127L-128LN A17L-A18R 131-132 128L-129R N A18R-A19L 132-133 129R-130L N A19L-A21L133-134 130L-131L N A21L-A20R 134-135 131L-132R N A20R-A22R 135-136132R-133R N A22R-A23R 136-137 133R-134R A23R-A24R 137-138 134R-135RA28L-A29L 141-142 139L-140L N A29L-A30L 142-143 140L-141L N

The orientations of these genes are variable, with some beingtranscribed to the right, some to the left. This means that some of theintergenic sites contain promoters that would have to be preserved inthe construction of the insertion vector. In addition, for overlappingconserved genes, during vector construction the genes would have to bereconstructed using alternative codons to minimize the repeatingsequences

The inventors focused on conserved genes whose orientation is “end toend” such that the 3′ stop codon of the genes are in close proximity toone another. The construction of transfer vectors used in these sitesare facilitated by the fact that there would be no promoter in thisregion between the stop codons. If there are intergenic nucleotidesseparating the stop codons, then construction of the insertion vector isstraightforward. If the stop codon of one gene is within the 3′ end ofthe other gene, then during construction of the plasmid transfer vector,the gene can be reconstructed using alternative codons to minimizerepeating sequences, or, depending on the size of the overlap, simplycorrected in the PCR of the flanks so as not to overlap. Table 2 givesthe intergenic sites that meet the requirement of the orientation of theconserved genes being “end to end”. Those intergenic sites highlightedin gray have no overlapping ends and therefore are simplest toconstruct.

TABLE 2 Conserved genes with “end to end” orientation

Gray highlighted genes have no overlappping ends and thus are simplestto use as intergenic sites.

From this list, the inventors focused on the six intergenic sites thathave no overlapping ends. In a working example, of these six, theintergenic site, 071-072 (I8R-G1L), was chosen as a site into which toinsert a heterologous gene. The construction of a recombinant MVA virususing this intergenic site, and the characteristics of the resultantvirus, are described in Example 1, and in International PublicationNumber WO2008/142479 A2, which is herein incorporated by reference inits entirety.

In addition to the conserved genes and corresponding intergenic sitesdescribed above, the inventors have discovered other sites useful forthe insertion of a heterologous nucleic acid sequence. For example, anygene, for which it has been experimentally demonstrated that thedeletion, or inactivation, of which, results in a 0.5 log, 0.75 log or 1log (10 fold) reduction in titer, could be considered an “essentialgene”. Similarly, an essential gene is any gene that results in at leastan 50%, at least a 75%, or at least a 90% reduction in titer compared toa virus in which the corresponding gene has not been deleted orinactivated. If this gene lies adjacent to another essential gene, theintergenic site between the two genes would be a useful site forinsertion of a heterologous nucleic acid sequence. While deletion of oneor more of these ORF, along with the intervening heterologous nucleicacid sequence, would not prevent the virus from growing, it would resultin decreased growth compared to a virus containing these ORFs. Thus,over time, virus that has lost one or more essential ORF would slowlybecome a smaller proportion of the total virus population and, givenenough time, would disappear from the virus population entirely.

Thus, one embodiment of the present invention is a recombinant modifiedvaccinia Ankara (MVA) virus comprising a heterologous nucleic acidsequence located between, or flanked by, two adjacent essential ORFsfrom MVA virus. In one embodiment, adjacent ORF's are separated by anintergenic region (IGR). As described, the IGR may contain aheterologous nucleic acid sequence. Thus, one embodiment is arecombinant modified vaccinia Ankara (MVA) virus comprising aheterologous nucleic acid sequence in an intergenic region locatedbetween, or flanked by, two adjacent essential ORFs from MVA virus.

As used herein, heterologous, or exogenous, nucleic acid sequences aresequences which, in nature, are not normally found associated with thepoxvirus as used according to the present invention. According to afurther embodiment of the present invention, the exogenous nucleic acidsequence comprises at least one coding sequence. The coding sequence isoperatively linked to a transcription control element, preferably to apoxviral transcription control element. Additionally, also combinationsbetween poxviral transcription control element and, e.g., internalribosomal entry sites can be used.

According to a further embodiment, the heterologous nucleic acidsequence can also comprise two or more coding sequences linked to one orseveral transcription control elements. Preferably, the coding sequenceencodes one or more proteins. In some embodiments, the proteins areantigens, or comprise antigenic epitopes, especially those oftherapeutically interesting genes.

Therapeutically interesting genes according to the present invention maybe genes derived from or homologous to genes of pathogenous orinfectious microorganisms which are disease causing. Accordingly, in thecontext of the present invention such therapeutically interesting genesare presented to the immune system of an organism in order to affect,preferably induce a specific immune response and, thereby, vaccinate orprophylactically protect the organism against an infection with themicroorganism. In further preferred embodiments of the present inventionthe therapeutically interesting genes are selected from genes ofinfectious viruses, e.g.,—but not limited to—dengue virus, hepatitisvirus B or C, or human immunodeficiency viruses such as HIV.

According to a preferred embodiment of the present invention theheterologous nucleic acid sequence is derived from HIV and encodes HIVenv, wherein the HIV env gene is preferably inserted into the IGRbetween the adjacent ORFs. The etiological agent of acquired immunedeficiency syndrome (AIDS) is recognized to be a retrovirus exhibitingcharacteristics typical of the lentivirus genus, referred to as humanimmunodeficiency virus (HIV). The phylogenetic relationships of thehuman lentiviruses are shown in FIG. 1. HIV-2 is more closely related toSIV_(smm), a virus isolated from sooty mangabey monkeys in the wild,than to HIV-1. It is currently believed that HIV-2 represents a zoonotictransmission of SIV_(smm) to man. A series of lentiviral isolates fromcaptive chimpanzees, designated SIV_(cpz), are close genetic relativesof HIV-1.

The earliest phylogenetic analyses of HIV-1 isolates focused on samplesfrom Europe/North America and Africa; discrete clusters of viruses wereidentified from these two areas of the world. Distinct genetic subtypesor clades of HIV-1 were subsequently defined and classified into threegroups: M (major); O (outlier); and N (non-M or O) (FIG. 2). The M groupof HIV-1, which includes over 95% of the global virus isolates, consistsof at least eight discrete clades (A, B, C, D, F, G, H, and J), based onthe sequence of complete viral genomes. Members of HIV-1 group O havebeen recovered from individuals living in Cameroon, Gabon, andEquatorial Guinea; their genomes share less than 50% identity innucleotide sequence with group M viruses. The more recently discoveredgroup N HIV-1 strains have been identified in infected Cameroonians,fail to react serologically in standard whole-virus enzyme-linkedimmunosorbent assay (ELISA), yet are readily detectable by conventionalWestern blot analysis.

Most current knowledge about HIV-1 genetic variation comes from studiesof group M viruses of diverse geographic origin. Data collected duringthe past decade indicate that the HIV-1 population present within aninfected individual can vary from 6% to 10% in nucleotide sequence.HIV-1 isolates within a clade may exhibit nucleotide distances of 15% ingag and up to 30% in gp120 coding sequences. Interclade geneticvariation may range between 30% and 40% depending on the gene analyzed.

All of the HIV-1 group M subtypes can be found in Africa. Clade Aviruses are genetically the most divergent and were the most commonHIV-1 subtype in Africa early in the epidemic. With the rapid spread ofHIV-1 to southern Africa during the mid to late 1990s, clade C viruseshave become the dominant subtype and now account for 48% of HIV-1infections worldwide. Clade B viruses, the most intensively studiedHIV-1 subtype, remain the most prevalent isolates in Europe and NorthAmerica.

High rates of genetic recombination are a hallmark of retroviruses. Itwas initially believed that simultaneous infections by geneticallydiverse virus strains were not likely to be established in individualsat risk for HIV-1. By 1995, however, it became apparent that asignificant fraction of the HIV-1 group M global diversity includedinterclade viral recombinants. It is now appreciated that HIV-1recombinants will be found in geographic areas such as Africa, SouthAmerica, and Southeast Asia, where multiple HIV-1 subtypes coexist andmay account for more than 10% of circulating HIV-1 strains. Molecularly,the genomes of these recombinant viruses resemble patchwork mosaics,with juxtaposed diverse HIV-1 subtype segments, reflecting the multiplecrossover events contributing to their generation. Most HIV-1recombinants have arisen in Africa and a majority contains segmentsoriginally derived from clade A viruses. In Thailand, for example, thecomposition of the predominant circulating strain consists of a clade Agag plus pol gene segment and a clade E env gene. Because the clade Eenv gene in Thai HIV-1 strains is closely related to the clade E envpresent in virus isolates from the Central African Republic, it isbelieved that the original recombination event occurred in Africa, withthe subsequent introduction of a descendent virus into Thailand.Interestingly, no full-length HIV-1 subtype E isolate (i.e., withsubtype E gag, pol, and env genes) has been reported to date.

The discovery that α and β chemokine receptors function as coreceptorsfor virus fusion and entry into susceptible CD4⁺ cells has led to arevised classification scheme for HIV-1 (FIG. 3). Isolates can now begrouped on the basis of chemokine receptor utilization in fusion assaysin which HIV-1 gp120 and CD4⁺ coreceptor proteins are expressed inseparate cells. As indicated in FIG. 3, HIV-1 isolates using the CXCR4receptor (now designated X4 viruses) are usually T cell line(TCL)-tropic syncytium inducing (SI) strains, whereas those exclusivelyutilizing the CCR5 receptor (R5 viruses) are predominantly macrophage(M)-tropic and non-syncytium inducing (NSI). The dual-tropic R5/X4strains, which may comprise the majority of patient isolates and exhibita continuum of tropic phenotypes, are frequently SI.

As is the case for all replication-competent retroviruses, the threeprimary HIV-1 translation products, all encoding structural proteins,are initially synthesized as polyprotein precursors, which aresubsequently processed by viral or cellular proteases into matureparticle-associated proteins (FIG. 4). The 55-kd Gag precursorPr55^(Gag) is cleaved into the matrix (MA), capsid (CA), nucleocapsid(NC), and p6 proteins. Autocatalysis of the 160-kd Gag-Pol polyprotein,Pr160^(Gag-Pol), gives rise to the protease (PR), the heterodimericreverse transcriptase (RT), and the integrase (IN) proteins, whereasproteolytic digestion by a cellular enzyme(s) converts the glycosylated160-kd Env precursor gp160 to the gp120 surface (SU) and gp41transmembrane (TM) cleavage products. The remaining six HIV-1-encodedproteins (Vif, Vpr, Tat, Rev, Vpu, and Nef) are the primary translationproducts of spliced mRNAs.

Gag

The Gag proteins of HIV, like those of other retroviruses, are necessaryand sufficient for the formation of noninfectious, virus-like particles.Retroviral Gag proteins are generally synthesized as polyproteinprecursors; the HIV-1 Gag precursor has been named, based on itsapparent molecular mass, Pr55^(Gag). As noted previously, the mRNA forPr55^(Gag) is the unspliced 9.2-kb transcript (FIG. 4) that requires Revfor its expression in the cytoplasm. When the pol ORF is present, theviral protease (PR) cleaves Pr55^(Gag) during or shortly after buddingfrom the cell to generate the mature Gag proteins p17 (MA), p24 (CA), p7(NC), and p6 (see FIG. 4). In the virion, MA is localized immediatelyinside the lipid bilayer of the viral envelope, CA forms the outerportion of the cone-shaped core structure in the center of the particle,and NC is present in the core in a ribonucleoprotein complex with theviral RNA genome (FIG. 5).

The HIV Pr55^(Gag) precursor oligomerizes following its translation andis targeted to the plasma membrane, where particles of sufficient sizeand density to be visible by EM are assembled. Formation of virus-likeparticles by Pr55^(Gag) is a self-assembly process, with criticalGag-Gag interactions taking place between multiple domains along the Gagprecursor. The assembly of virus-like particles does not require theparticipation of genomic RNA (although the presence of nucleic acidappears to be essential), pol-encoded enzymes, or Env glycoproteins, butthe production of infectious virions requires the encapsidation of theviral RNA genome and the incorporation of the Env glycoproteins and theGag-Pol polyprotein precursor Pr160^(Gag-Pol).

Pol

Downstream of gag lies the most highly conserved region of the HIVgenome, the pol gene, which encodes three enzymes: PR, RT, and IN (seeFIG. 4). RT and IN are required, respectively, for reverse transcriptionof the viral RNA genome to a double-stranded DNA copy, and for theintegration of the viral DNA into the host cell chromosome. PR plays acritical role late in the life cycle by mediating the production ofmature, infectious virions. The pol gene products are derived byenzymatic cleavage of a 160-kd Gag-Pol fusion protein, referred to asPr160^(Gag-Pol). This fusion protein is produced by ribosomalframeshifting during translation of Pr55^(Gag) (see FIG. 4). Theframe-shifting mechanism for Gag-Pol expression, also utilized by manyother retroviruses, ensures that the pal-derived proteins are expressedat a low level, approximately 5% to 10% that of Gag. Like Pr55^(Gag),the N-terminus of Pr160^(Gag-Pol) is myristylated and targeted to theplasma membrane.

Protease

Early pulse-chase studies performed with avian retroviruses clearlyindicated that retroviral Gag proteins are initially synthesized aspolyprotein precursors that are cleaved to generate smaller products.Subsequent studies demonstrated that the processing function is providedby a viral rather than a cellular enzyme, and that proteolytic digestionof the Gag and Gag-Pol precursors is essential for virus infectivity.Sequence analysis of retroviral PRs indicated that they are related tocellular “aspartic” proteases such as pepsin and renin. Like thesecellular enzymes, retroviral PRs use two apposed Asp residues at theactive site to coordinate a water molecule that catalyzes the hydrolysisof a peptide bond in the target protein. Unlike the cellular asparticproteases, which function as pseudodimers (using two folds within thesame molecule to generate the active site), retroviral PRs function astrue dimers. X-ray crystallographic data from HIV-1 PR indicate that thetwo monomers are held together in part by a four-stranded antiparallelβ-sheet derived from both N- and C-terminal ends of each monomer. Thesubstrate-binding site is located within a cleft formed between the twomonomers. Like their cellular homologs, the HIV PR dimer containsflexible “flaps” that overhang the binding site and may stabilize thesubstrate within the cleft; the active-site Asp residues lie in thecenter of the dimer. Interestingly, although some limited amino acidhomology is observed surrounding active-site residues, the primarysequences of retroviral PRs are highly divergent, yet their structuresare remarkably similar.

Reverse Transcriptase

By definition, retroviruses possess the ability to convert theirsingle-stranded RNA genomes into double-stranded DNA during the earlystages of the infection process. The enzyme that catalyzes this reactionis RT, in conjunction with its associated RNaseH activity. RetroviralRTs have three enzymatic activities: (a) RNA-directed DNA polymerization(for minus-strand DNA synthesis), (b) RNaseH activity (for thedegradation of the tRNA primer and genomic RNA present in DNA-RNA hybridintermediates), and (c) DNA-directed DNA polymerization (for second- orplus-strand DNA synthesis).

The mature HIV-1 RT holoenzyme is a heterodimer of 66 and 51 kdsubunits. The 51-kd subunit (p51) is derived from the 66-kd (p66)subunit by proteolytic removal of the C-terminal 15-kd RNaseH domain ofp66 by PR (see FIG. 4). The crystal structure of HIV-1 RT reveals ahighly asymmetric folding in which the orientations of the p66 and p51subunits differ substantially. The p66 subunit can be visualized as aright hand, with the polymerase active site within the palm, and a deeptemplate-binding cleft formed by the palm, fingers, and thumbsubdomains. The polymerase domain is linked to RNaseH by the connectionsubdomain. The active site, located in the palm, contains three criticalAsp residues (110, 185, and 186) in close proximity, and two coordinatedMg²⁺ ions. Mutation of these Asp residues abolishes RT polymerizingactivity. The orientation of the three active-site Asp residues issimilar to that observed in other DNA polymerases (e.g., the Klenowfragment of E. coli DNA poll). The p51 subunit appears to be rigid anddoes not form a polymerizing cleft; Asp 110, 185, and 186 of thissubunit are buried within the molecule. Approximately 18 base pairs ofthe primer-template duplex lie in the nucleic acid binding cleft,stretching from the polymerase active site to the RNaseH domain.

In the RT-primer-template-dNTP structure, the presence of adideoxynucleotide at the 3′ end of the primer allows visualization ofthe catalytic complex trapped just prior to attack on the incoming dNTP.Comparison with previously obtained structures suggests a model wherebythe fingers close in to trap the template and dNTP prior to nucleophilicattack of the 3′-OH of the primer on the incoming dNTP. After theaddition of the incoming dNTP to the growing chain, it has been proposedthat the fingers adopt a more open configuration, thereby releasing thepyrophosphate and enabling RT to bind the next dNTP. The structure ofthe HIV-1 RNaseH has also been determined by x-ray crystallography; thisdomain displays a global folding similar to that of E. coli RNaseH.

Integrase

A distinguishing feature of retrovirus replication is the insertion of aDNA copy of the viral genome into the host cell chromosome followingreverse transcription. The integrated viral DNA (the provirus) serves asthe template for the synthesis of viral RNAs and is maintained as partof the host cell genome for the lifetime of the infected cell.Retroviral mutants deficient in the ability to integrate generally failto establish a productive infection.

The integration of viral DNA is catalyzed by integrase, a 32-kd proteingenerated by PR-mediated cleavage of the C-terminal portion of the HIV-1Gag-Pol polyprotein (see FIG. 4).

Retroviral IN proteins are composed of three structurally andfunctionally distinct domains: an N-terminal, zinc-finger-containingdomain, a core domain, and a relatively nonconserved C-terminal domain.Because of its low solubility, it has not yet been possible tocrystallize the entire 288-amino-acid HIV-1 IN protein. However, thestructure of all three domains has been solved independently by x-raycrystallography or NMR methods. The crystal structure of the core domainof the avian sarcoma virus IN has also been determined. The N-terminaldomain (residues 1 to 55), whose structure was solved by NMRspectroscopy, is composed of four helices with a zinc coordinated byamino acids His-12, His-16, Cys-40, and Cys-43. The structure of theN-terminal domain is reminiscent of helical DNA binding proteins thatcontain a so-called helix-turn-helix motif; however, in the HIV-1structure this motif contributes to dimer formation. Initially, poorsolubility hampered efforts to solve the structure of the core domain.However, attempts at crystallography were successful when it wasobserved that a Phe-to-Lys change at IN residue 185 greatly increasedsolubility without disrupting in vitro catalytic activity. Each monomerof the HIV-1 IN core domain (IN residues 50 to 212) is composed of afive-stranded β-sheet flanked by helices; this structure bears strikingresemblance to other polynucleotidyl transferases including RNaseH andthe bacteriophage MuA transposase. Three highly conserved residues arefound in analogous positions in other polynucleotidyl transferases; inHIV-1 IN these are Asp-64, Asp-116 and Glu-152, the so-called D,D-35-Emotif. Mutations at these positions block HIV IN function both in vivoand in vitro. The close proximity of these three amino acids in thecrystal structure of both avian sarcoma virus and HIV-1 core domainssupports the hypothesis that these residues play a central role incatalysis of the polynucleotidyl transfer reaction that is at the heartof the integration process. The C-terminal domain, whose structure hasbeen solved by NMR methods, adopts a five-stranded β-barrel foldingtopology reminiscent of a Src homology 3 (SH3) domain. Recently, thex-ray structures of SIV and Rous sarcoma virus IN protein fragmentsencompassing both the core and C-terminal domains have been solved.

Env

The HIV Env glycoproteins play a major role in the virus life cycle.They contain the determinants that interact with the CD4 receptor andcoreceptor, and they catalyze the fusion reaction between the lipidbilayer of the viral envelope and the host cell plasma membrane. Inaddition, the HIV Env glycoproteins contain epitopes that elicit immuneresponses that are important from both diagnostic and vaccinedevelopment perspectives.

The HIV Env glycoprotein is synthesized from the singly spliced 4.3-kbVpu/Env bicistronic mRNA (see FIG. 4); translation occurs on ribosomesassociated with the rough endoplasmic reticulum (ER). The 160-kdpolyprotein precursor (gp160) is an integral membrane protein that isanchored to cell membranes by a hydrophobic stop-transfer signal in thedomain destined to be the mature TM Env glycoprotein, gp41 (FIG. 6). Thegp160 is cotranslationally glycosylated, forms disulfide bonds, andundergoes oligomerization in the ER. The predominant oligomeric formappears to be a trimer, although dimers and tetramers are also observed.The gp160 is transported to the Golgi, where, like other retroviralenvelope precursor proteins, it is proteolytically cleaved by cellularenzymes to the mature SU glycoprotein gp120 and TM glycoprotein gp41(see FIG. 6). The cellular enzyme responsible for cleavage of retroviralEnv precursors following a highly conserved Lys/Arg-X-Lys/Arg-Arg motifis furin or a furin-like protease, although other enzymes may alsocatalyze gp160 processing. Cleavage of gp160 is required for Env-inducedfusion activity and virus infectivity. Subsequent to gp160 cleavage,gp120 and gp41 form a noncovalent association that is critical fortransport of the Env complex from the Golgi to the cell surface. Thegp120-gp41 interaction is fairly weak, and a substantial amount of gp120is shed from the surface of Env-expressing cells.

The HIV Env glycoprotein complex, in particular the SU (gp120) domain,is very heavily glycosylated; approximately half the molecular mass ofgp160 is composed of oligosaccharide side chains. During transport ofEnv from its site of synthesis in the ER to the plasma membrane, many ofthe side chains are modified by the addition of complex sugars. Thenumerous oligosaccharide side chains form what could be imagined as asugar cloud obscuring much of gp120 from host immune recognition. Asshown in FIG. 6, gp120 contains interspersed conserved (C₁ to C₅) andvariable (V₁ to V₅) domains. The Cys residues present in the gp120s ofdifferent isolates are highly conserved and form disulfide bonds thatlink the first four variable regions in large loops.

A primary function of viral Env glycoproteins is to promote a membranefusion reaction between the lipid bilayers of the viral envelope andhost cell membranes. This membrane fusion event enables the viral coreto gain entry into the host cell cytoplasm. A number of regions in bothgp120 and gp41 have been implicated, directly or indirectly, inEnv-mediated membrane fusion. Studies of the HA₂ hemagglutinin proteinof the orthomyxoviruses and the F protein of the paramyxovirusesindicated that a highly hydrophobic domain at the N-terminus of theseproteins, referred to as the fusion peptide, plays a critical role inmembrane fusion. Mutational analyses demonstrated that an analogousdomain was located at the N-terminus of the HIV-1, HIV-2, and SIV TMglycoproteins (see FIG. 6). Nonhydrophobic substitutions within thisregion of gp41 greatly reduced or blocked syncytium formation andresulted in the production of noninfectious progeny virions.

C-terminal to the gp41 fusion peptide are two amphipathic helicaldomains (see FIG. 6) which play a central role in membrane fusion.Mutations in the N-terminal helix (referred to as the N-helix), whichcontains a Leu zipper-like heptad repeat motif, impair infectivity andmembrane fusion activity, and peptides derived from these sequencesexhibit potent antiviral activity in culture. The structure of theectodomain of HIV-1 and SIV gp41, the two helical motifs in particular,has been the focus of structural analyses in recent years. Structureswere determined by x-ray crystallography or NMR spectroscopy either forfusion proteins containing the helical domains, a mixture of peptidesderived from the N- and C-helices, or in the case of the SIV structure,the intact gp41 ectodomain sequence from residue 27 to 149. Thesestudies obtained fundamentally similar trimeric structures, in which thetwo helical domains pack in an antiparallel fashion to generate asix-helix bundle. The N-helices form a coiled-coil in the center of thebundle, with the C-helices packing into hydrophobic grooves on theoutside.

In the steps leading to membrane fusion CD4 binding induces conformationchanges in Env that facilitate coreceptor binding. Following theformation of a ternary gp120/CD4/coreceptor complex, gp41 adopts ahypothetical conformation that allows the fusion peptide to insert intothe target lipid bilayer. The formation of the gp41 six-helix bundle(which involves antiparallel interactions between the gp41 N- andC-helices) brings the viral and cellular membranes together and membranefusion takes place.

Furthermore, therapeutically interesting genes according to the presentinvention also comprise disease related genes, which have a therapeuticeffect on proliferative disorder, cancer or metabolic diseases. Forexample, a therapeutically interesting gene regarding cancer could be acancer antigen that has the capacity to induce a specific anti-cancerimmune reaction.

According to a further embodiment of the present invention, theheterologous nucleic acid sequence comprises at least one marker orselection gene.

Selection genes transduce a particular resistance to a cell, whereby acertain selection method becomes possible. The skilled practitioner isfamiliar with a variety of selection genes, which can be used in apoxviral system. Among these are, e.g., neomycin resistance gene (NPT)or phosphoribosyl transferase gene (gpt).

Marker genes induce a color reaction in transduced cells, which can beused to identify transduced cells. The skilled practitioner is familiarwith a variety of marker genes, which can be used in a poxviral system.Among these are the gene encoding, e.g., β-galactosidase (β-gal),β-glucosidase (β-glu), green fluorescence protein (EGFP) or bluefluorescence protein.

According to still a further embodiment of the present invention theheterologous nucleic acid sequence comprises a spacing sequence, whichseparates poxviral transcription control element and/or coding sequencein the heterologous nucleic acid sequence from the stop codon and/or thestart codon of the adjacent ORFs. This spacer sequence between thestop/start codon of the adjacent ORF and the inserted coding sequence inthe heterologous nucleic acid sequence has the advantage to stabilizethe inserted heterologous nucleic acid sequence and, thus, any resultingrecombinant virus. The size of the spacer sequence is variable as longas the sequence is without its own coding or regulatory function.

According to a further embodiment, the spacer sequence separating thepoxviral transcription control element and/or the coding sequence in theheterologous nucleic acid sequence from the stop codon of the adjacentORF is at least one nucleotide long.

According to another embodiment of the present invention, the spacingsequence separating the poxviral transcription control element and/orthe coding sequence in the heterologous nucleic acid sequence from thestart codon of the adjacent ORF is at least 30 nucleotides.Particularly, in cases where a typical vaccinia virus promoter elementis identified upstream of a start codon the insertion of heterologousnucleic acid sequence may not separate the promoter element from thestart codon of the adjacent ORF. A typical vaccinia promoter element canbe identified by scanning for e.g., the sequence “TAAAT” for latepromoters (Davison & Moss 1989 J. Mol. Biol.; 210:771-784) and an A/Trich domain for early promoters. A spacing sequence of about 30nucleotides is the preferred distance to secure that a poxviral promoterlocated upstream of the start codon of the ORF is not influenced.Additionally, according to a further preferred embodiment, the distancebetween the inserted heterologous nucleic acid sequence and the startcodon of the adjacent ORF is around 50 nucleotides and more preferablyaround 100 nucleotides.

According to a further preferred embodiment of the present invention,the spacing sequence comprises an additional poxviral transcriptioncontrol element which is capable pf controlling the transcription of theadjacent ORF.

Thus far, the disclosure has focused on recombinant MVA viruses usingORFs that are adjacent in parental MVA virus. However, the presentinvention also includes recombinant viruses, and methods of making suchviruses, in which heterologous nucleic acid sequences are insertedbetween adjacent, essential ORFs techniques, wherein the ORfs used forinsertion are not adjacent in the parental MVA virus. That is, virusescan be constructed so that ORFs that are adjacent in the recombinant MVAvirus are separated by one or more poxvirus ORFs (intervening ORFs) inthe parental MVA virus. As used herein, a parental MVA virus is one fromwhich a progeny, recombinant virus is constructed. An example of aparental MVA virus is MVA 1974/NIH Clone 1. Parental viruses can be usedto construct recombinant viruses using techniques disclosed herein, suchthat the intervening ORFs can be removed during the constructionprocess. It is appreciated by those skilled in the poxvirus arts that byusing nucleic acid molecules comprising carefully selected poxvirusORF's, sections of the viral genome between those two ORFs can bedeleted through the process of homologous recombination. For example, itcan be supposed that two essential ORFs are separated by a one kilobaseregion of the genome containing a non-essential ORF. A nucleic acidconstruct can be made in which the two essential ORFs are cloned, forexample, into a plasmid such that the two ORFs are adjacent in thenucleic acid construct. Upon introduction of the nucleic acid constructinto a poxvirus infected cell (e.g., a parental MVA virus infectedcell), the essential ORFs will recombine with the corresponding ORFs inthe viral genome of the parental virus. Through further recombinationevents understood by those skilled in the art, the one kilobase regionwill be excised from the viral genome, resulting in the two essentialORF becoming adjacent. Thus, one embodiment of the present invention isa recombinant modified vaccinia Ankara (MVA) virus comprising aheterologous nucleic acid sequence located between two adjacentessential ORFs from the MVA virus genome, wherein the recombinant MVAvirus lacks non-essential ORFs that are present between thecorresponding essential ORFs in the parental MVA virus. Thus theheterologous nucleic acid sequence is flanked by essential ORFs that arenon-adjacent in the parental MVA virus. The essential ORF are chosenfrom pairs of essential ORFs present in the MVA genome that areseparated by non-essential ORFs. In one embodiment, the essential ORFsare selected from the group consisting of A50R (MVA163), B1R (MVA167),F10 (MVA-039), F12 (MVA042), F13L (MVA043), F15L (MVA045), F17L(MVA047), E4L (MVA051), E6L (MVA053), E8L (MVA055), E10L (MVA057), I1L(MVA062), I3L (MVA064), I5L (MVA066), J1R (MVA085), J3R (MVA087), D7L(MVA104), D9L (MVA106), A24R (MVA135), and A28R (MVA139). In oneembodiment, the two essential ORFs are selected from pairs of essentialORFs in the group of consisting of A50R-B1R (MVA163-MVA167), F10-F12(MVA039-MVA042), F13L-F15L (MVA043-MVA045), F15L-F17L (MVA045-MVA047),E4L-E6L (MVA051-MVA053), E6L-E8L (MVA053-MVA055), E10L-I1L(MVA057-MVA062), I3L-I5L (MVA064-MVA066), J1R-J3R (MVA085-MVA087),D7L-D9L (MVA104-MVA106), and A24R-A28R (MVA135-MVA139). In oneembodiment, the essential ORFs are selected from A50R (MVA163) and B1R(MVA167). In one embodiment, one essential ORF is A50R (MVA163) and theother essential ORF is B1R (MVA167).

As previously discussed, as a result of extensive passage in cellculture, the MVA virus genome contains six major deletions, referred toas Del I, II, II, IV, V and VI. Historically, the region around Del III,which is a deletion of approximately 31,000 nucleotides, has been usedfor insertion of heterologous nucleic acid sequences. Thus, in oneembodiment of the present invention, the non-essential ORFs deletedduring construction of the recombinant MVA virus flank the Del IIIregion in the wild-type MVA virus.

As has been described, recombinant MVA viruses can contain additionalsequences, such as IGRs and/or heterologous nucleic acid sequences,between the two adjacent, essential ORFs. Such sequences have beendescribed herein. Thus, one embodiment of the present invention is arecombinant modified vaccinia Ankara (MVA) virus comprising aheterologous nucleic acid sequence located between two adjacentessential ORFs from the MVA virus genome, wherein the recombinant MVAvirus lacks non-essential ORFs that are present between thecorresponding essential ORFs in the parental MVA virus, and wherein theheterologous nucleic acid sequence is inserted into an IGR. Theheterologous can contain coding sequences under the control of atranscriptional control element, as has been described elsewhere in thedisclosure.

While the inventors have disclosed specific essential ORFs, andsequences thereof, the present invention also comprises recombinant MVAvirus, and methods of making such, using portions or variants of thedisclosed ORFs. For example, while the present invention discloses ORFA50R, and portions thereof, (SEQ ID NO:11 and SEQ ID NO:14), and ORFB1R, and portions thereof (SEQ ID NO:16 and SEQ ID NO:19), the presentinvention comprises recombinant MVA viruses comprising variants of thesesequences, so long as the variant ORF encodes a protein havingessentially the same function as the protein encoded by thecorresponding wild-type ORF. Two proteins are considered as havingessentially the same function if MVA viruses comprising the respectiveproteins produce titers that are within about 10%, about 20%, about 30%or about 40% of each other when grown using the same cell line. Thus,one embodiment of the present invention is a recombinant modifiedvaccinia Ankara (MVA) virus comprising a heterologous nucleic acidsequence located between two adjacent ORFs, wherein the adjacent ORFscomprise a nucleotide sequence at least 90%, at least 95%, at least 97%or at least 99% sequence identity with an essential ORF from MVA. In oneembodiment, the adjacent ORFs comprise a nucleotide sequence at least90%, at least 95%, at least 97% or at least 99% identical to essentialORFs selected from the group consisting of A50R (MVA163), B1R (MVA167),F10 (MVA-039), F12 (MVA042), F13L (MVA043), F15L (MVA045), F17L(MVA047), E4L (MVA051), E6L (MVA053), E8L (MVA055), E10L (MVA057), I1L(MVA062), I3L (MVA064), I5L (MVA066), J1R (MVA085), J3R (MVA087), D7L(MVA104), D9L (MVA106), A24R (MVA135), and A28R (MVA139). In a preferredembodiment, the two adjacent ORf's are not derived from the sameessential ORF. In one embodiment, the two adjacent ORFs comprisenucleotide sequences at least 90%, at least 95%, at least 97% or atleast 99% identical to pairs of essential ORFs in the group ofconsisting of A50R-B1R (MVA163-MVA167), F10-F12 (MVA039-MVA042),F13L-F15L (MVA043-MVA045), F15L-F17L (MVA045-MVA047), E4L-E6L(MVA051-MVA053), E6L-E8L (MVA053-MVA055), E10L-I1L (MVA057-MVA062),I3L-I5L (MVA064-MVA066), J1R-J3R (MVA085-MVA087), D7L-D9L(MVA104-MVA106), and A24R-A28R (MVA135-MVA139). In one embodiment oneadjacent ORF comprises a nucleotide sequence at least 90%, at least 95%,at least 97% or at least 99% sequence identical with SEQ ID NO:A50R(MVA163) and the second adjacent ORF comprises a nucleotide sequence atleast 90%, at least 95%, at least 97% or at least 99% sequence identicalto a second essential ORF. In one embodiment one adjacent ORF comprisesa nucleotide sequence at least 90%, at least 95%, at least 97% or atleast 99% sequence identical with SEQ ID NO:B1R.

The present invention also discloses nucleic acid constructs useful forproducing recombinant viruses of the present invention. As used herein anucleic acid construct is a recombinant nucleic acid molecule comprisingat least a portion of at least one essential ORF from MVA virus. Thenucleic acid construct enables transport of useful nucleic acidsequences to a cell within an environment, such as, but not limited to,an organism, tissue, or cell culture. A nucleic acid construct of thepresent disclosure is produced by human intervention. The nucleic acidconstruct can be DNA, RNA or variants thereof. The nucleic acid moleculecan be linear DNA, a DNA plasmid, a viral vector, or other vector. Inone embodiment, a nucleic acid molecule can be a DNA plasmid. In oneembodiment, a nucleic acid molecule can be a DNA plasmid comprisingviral components, plasmid components, transcriptional control elements,and any other useful elements know to those skilled in the art thatenable nucleic acid molecule delivery and expression. Methods for thegeneral construction of recombinant nucleic acid molecules are wellknown. See, for example, Molecular Cloning: a Laboratory Manual, 3^(rd)edition, Sambrook et al. 2001 Cold Spring Harbor Laboratory Press, andCurrent Protocols in Molecular Biology, Ausubel et al. eds., John Wiley& Sons, 1994.

One embodiment of the present invention is an isolated nucleic acidconstruct comprising: (a) a first nucleic acid sequence derived from, orhomologous to, a first essential ORF from a modified vaccinia Ankara(MVA) virus genome; and (b) a second nucleic acid sequence derived from,or homologous to, a second essential ORF from a MVA virus genome;wherein the first and second essential MVA virus ORFs are separated byat least one non-essential ORF in the MVA virus genome, and wherein thefirst and second nucleic acid sequences are adjacent to each other inthe isolated nucleic acid construct, and wherein the first and secondnucleic acid sequences comprise at least 25 contiguous nucleotides fromthe first and second essential MVA ORFs, respectively. Such a nucleicacid construct is useful for constructing recombinant MVA virusesthrough the process of homologous recombination. Using this process,isolated nucleic acid constructs of the present invention can be used toconstruct recombinant MVA viruses in which ORFs that are not adjacent ina parental MVA virus (i.e, they are separated by other, non-essentialMVA ORFs), are made adjacent in the progeny, recombinant MVA virus. Thiscan be done, for example, by cloning non-adjacent ORFs from a parentalMVA virus into a nucleic acid molecule, such as a plasmid, without alsocloning the intervening non-essential ORFs. Thus, the no-adjacent ORFsare made adjacent in the nucleic acid construct. As has been described,recombination of such a nucleic acid construct into the MVA viral genomewill result in deletion of the intervening non-essential ORFs from theparental MVA virus resulting in a progeny, recombinant MVA virus inwhich the originally non-adjacent ORFs are adjacent. Thus, in apreferred embodiment, the first and second nucleic acid sequences arederived from, or homologous to, first and second essential MVA ORFs,respectively, that are not adjacent in the parental MVA virus. That is,the first and second essential ORFs are separated by at least onenon-essential ORF in the parental MVA virus genome.

As used herein, the phrase derived from refers to the source nucleicacid (i.e., ORF) from which the nucleic acid sequence was obtained.Thus, in this regard the nucleic acid sequence may be identical to allor part of the originating ORF. However, the nucleic acid sequence mayalso vary in sequence from the originating ORF. Thus, a nucleic acidsequence that is derived from an MVA ORF may or may not be identical insequence to all, or a portion, of an MVA ORF, so long as the function ofthe original ORF is maintained in the derived nucleic acid sequence. Forexample, it is understood in the art that nucleic acid molecules fromrelated species of poxviruses can recombine, even though the sequencesof such molecules are not identical. Thus, in one embodiment of thepresent invention, the first and second nucleic acid sequences havesufficient sequence identity with the essential MVA ORFs from which theyarea derived to allow homologous recombination between a nucleic acidmolecule comprising the first or second nucleic acid sequence, and anucleic acid molecule comprising the essential MVA ORF from which suchsequence was derived. In one embodiment, the first and second nucleicacid sequences are at least 75%, at least 85%, at least 90%, at least95%, at least 97%, or at least 99% identical to at least a portion ofthe essential MVA ORF from which they are derived. In one embodiment,the nucleic acid sequence is identical to at least a portion of theessential MVA ORF from which it was derived.

It is also appreciated in the art that small polynucleotide moleculesare capable of engaging in the process of homologous recombination.Consequently, nucleic acid sequences present in nucleic acid constructsof the present invention need not comprise the entire sequence of anessential MVA ORF in order for the nucleic acid construct to be able torecombine into the MVA virus genome. In fact, it has been shown thatfragments of the poxvirus genome as small as 20 bases in length arecapable of engaging in homologous recombination with their respectivesequence in the viral genome. Thus, in one embodiment of the presentinvention, the first and second nucleic acid sequences can comprise 25,30, 35, 40, 45, 50, 100, 150, 200, 250, or 300 nucleotides from anessential MVA ORF. One embodiment of the present invention is anisolated nucleic acid construct comprising: (a) a first nucleic acidsequence comprising at least 25 contiguous nucleotides from a firstessential MVA ORF; and (b) a second nucleic acid sequence comprising atleast 25 contiguous nucleotides from a second essential MVA ORF; whereinthe first and second essential MVA virus ORFs are separated by at leastone non-essential ORF in the MVA virus genome, and wherein the first andsecond nucleic acid sequences are adjacent to each other in the isolatednucleic acid construct. In one embodiment, the first nucleic acidsequences comprise 25 contiguous nucleotides from an essential ORFselected from the group consisting of A50R (MVA163), B1R (MVA167), F10(MVA-039), F12 (MVA042), F13L (MVA043), F15L (MVA045), F17L (MVA047),E4L (MVA051), E6L (MVA053), E8L (MVA055), E10L (MVA057), I1L (MVA062),I3L (MVA064), I5L (MVA066), J1R (MVA085), J3R (MVA087), D7L (MVA104),D9L (MVA106), A24R (MVA135), and A28R (MVA139). In one embodiment, thesecond nucleic acid sequences comprise 25 contiguous nucleotides from anessential ORF selected from the group consisting of A50R (MVA163), B1R(MVA167), F10 (MVA-039), F12 (MVA042), F13L (MVA043), F15L (MVA045),F17L (MVA047), E4L (MVA051), E6L (MVA053), E8L (MVA055), E10L (MVA057),I1L (MVA062), I3L (MVA064), I5L (MVA066), J1R (MVA085), J3R (MVA087),D7L (MVA104), D9L (MVA106), A24R (MVA135), and A28R (MVA139). In oneembodiment, the first nucleic acid sequence comprises at least 25contiguous nucleotides from SEQ ID NO:11 or SEQ ID NO:14, and the secondnucleic acid sequence comprises at least 25 contiguous nucleotides fromSEQ ID NO:16 or SEQ ID NO:19.

Nucleic acid constructs of the present invention are used to deliverheterologous nucleic acid sequences into the genome of MVA virus. Thus,one embodiment, a nucleic acid construct of the present inventioncomprises a heterologous nucleic acid molecule between the first andsecond nucleic acid sequences. Exemplary heterologous nucleic acidsequences have been described elsewhere in the disclosure. Anyheterologous nucleic acid sequence disclosed herein is suitable forinclusion in a nucleic acid construct of the present invention.

Because nucleic acid constructs of the present invention can recombinewith the genome of a parental MVA virus, they can be used to insertheterologous nucleic acid sequences into the viral genome. Thus, in oneembodiment of the present invention a nucleic acid contrast of thepresent invention contains an intergenic region between the first andsecond nucleic acid sequences. The intergenic region can comprise suchthings as transcriptional control elements, restriction sites andnon-vaccinia open reading frames. Thus, the intergenic region can beused to insert heterologous nucleic acid sequences comprising genesunder the control of a transcriptional control element. Uponrecombination of the nucleic acid construct with the MVA virus genome,the heterologous nucleic acid sequence will be inserted into the MVAviral genome between the essential ORFs corresponding to the twoadjacent, essential ORFs flanking the nucleic acid sequence in thenucleic acid construct. The resulting MVA virus will be a recombinantMVA virus containing the heterologous nucleic acid sequence stablyintegrated into the MVA virus genome.

In one embodiment, a nucleic acid construct of the present inventioncomprises complete or partial fragment of an IGR sequence locatedbetween the two adjacent ORFs of the viral genome. Preferably, thenucleic acid construct comprises inserted into said IGR-derived sequenceat least one cloning site for the insertion of an heterologous DNAsequence of interest and, preferably, for the insertion of a poxviraltranscription control element operatively linked to said heterologousDNA sequence. Optionally, the nucleic acid construct comprises areporter- and/or selection gene cassette. The nucleic acid constructpreferably also comprises sequences of the two adjacent ORFs flankingsaid complete or partial fragment of the IGR sequence.

Some IGRs have been identified which do not include nucleotidesequences. In these cases, the plasmid vector comprises DNA sequences ofthe IGR flanking sequences, i.e., DNA sequences of the two adjacentORFs. Preferably, the cloning site for the insertion of the heterologousDNA sequence is inserted into the IGR. The DNA of the IGR flankingsequences is used to direct the insertion of exogenous DNA sequencesinto the corresponding IGR in the MVA genome. Such a plasmid vector mayadditionally include a complete or partial fragment of an IGR sequencewhich comprises the cloning site for the insertion of the heterologousDNA sequence and, optionally, of the, reporter- and/or selection genecassette.

One embodiment of the present invention is a method to produce a stable,recombinant modified vaccinia Ankara virus. Such a method makes use ofthe nucleic acid constructs disclosed herein. Thus, the method comprisesfirst obtaining a nucleic acid construct comprising a heterologousnucleic acid sequence located between, or flanked by, two adjacentessential open reading frames (ORFs) of the MVA virus genome, whereinthe MVA virus is lacking non-essential ORFS, or ORF fragments, that arepresent between the corresponding two essential ORFS in the parental MVAvirus. For example, to obtain an appropriate nucleic acid construct,nucleic acid sequences from essential MVA ORFs can be isolated andcloned into a standard cloning vector, such as pBluescript (Stratagene),so that they flank the heterologous DNA to be inserted into the MVAgenome. This construct can then be introduced into a cell using methodsknow to those in the art (e.g., transfection). The cell containing thenucleic acid construct is then infected with a MVA virus and culturedunder conditions suitable to allow homologous recombination between thenucleic acid construct and the MVA virus genome. At the appropriate timethe cells are then harvested and the recombinant MVA virus isolated. Theresultant virus will be a stable, recombinant MVA virus. Such a virusmay also be called a derivative virus. It will be appreciated that theorder of the steps of introducing the nucleic acid construct into thecell, and infecting the cell can be reversed, or that these two stepsmay happen simultaneously.

General methods to introduce heterologous nucleic acid sequences in anucleic acid construct into an MVA genome and methods to obtainrecombinant MVA are well known to the person skilled in the art and,additionally, can be deduced can be deduced from Molecular Cloning, ALaboratory Manual, Second Edition, J. Sambrook, E. F. Fritsch and T.Maniatis, Cold Spring Harbor Laboratory Press, 1989 and CurrentProtocols in Molecular Biology, John Wiley and Son Inc. 1998, Chapter16, section IV, “Expression of proteins in mammalian cells usingvaccinia viral vectors”.

The DNA sequences according to the invention can be used to identify orisolate the MVA or its derivatives according to the invention and cellsor individuals infected with an MVA according to the present invention.The DNA sequences are, e.g., used to generate PCR-primers, hybridizationprobes or are used in array technologies.

The term derivative virus, and the like, according to the presentinvention refers to progeny viruses showing the same characteristicfeatures as the parent virus but showing differences in one or moreparts of its genome. The term “derivative of MVA” describes a virus,which has the same functional characteristics compared to MVA. Forexample, a derivative of MVA 1974/NIH Clone 1 has the characteristicfeatures of MVA 1974/NIH Clone 1. One of these characteristics of MVA1974/NIH Clone for derivatives thereof is its attenuation and severerestriction in host range.

The recombinant MVA according to the present invention is useful as amedicament or vaccine. Thus, one embodiment of the present invention isa method to protect an individual from a disease using a recombinant MVAvirus of the present invention.

A recombinant MVA virus of the present invention can also be used forthe introduction of the exogenous coding sequence into a target cell,said sequence being either homologous or heterologous to the genome ofthe target cell. The introduction of an exogenous coding sequence into atarget cell may be done in vitro to produce proteins, polypeptides,peptides, antigens or antigenic epitopes. This method comprises theinfection of a host cell with the recombinant MVA according to theinvention, cultivation of the infected host cell under suitableconditions, and isolation and/or enrichment of the polypeptide, peptide,protein, antigen, epitope and/or virus produced by said host cell.

Furthermore, the method for introduction of one or more homologous orone or more heterologous sequence into cells may be applied for in vitroand in vivo therapy. For in vitro therapy, isolated cells that have beenpreviously (ex vivo) infected with the recombinant MVA according to theinvention are administered to the living animal body for affecting,preferably inducing an immune response. For in vivo therapy, therecombinant poxvirus according to the invention is directly administeredto the living animal body for affecting, preferably inducing an immuneresponse. In this case, the cells surrounding the site of inoculation,but also cells where the virus is transported to via, e.g., the bloodstream, are directly infected in vivo by the recombinant MVA accordingto the invention. After infection, these cells synthesize the proteins,peptides or antigenic epitopes of the therapeutic genes, which areencoded by the exogenous coding sequences and, subsequently, presentthem or parts thereof on the cellular surface. Specialized cells of theimmune system recognize the presentation of such heterologous proteins,peptides or epitopes and launch a specific immune response.

Since the MVA is highly growth restricted and, thus, highly attenuated,it is useful for the treatment of a wide range of mammals includinghumans, including immune-compromised animals or humans. The presentinvention also provides pharmaceutical compositions and vaccines forinducing an immune response in a living animal body, including a human.

The pharmaceutical composition may generally include one or morepharmaceutical acceptable and/or approved carriers, additives,antibiotics, preservatives, adjuvants, diluents and/or stabilizers. Suchauxiliary substances can be water, saline, glycerol, ethanol, wetting oremulsifying agents, pH buffering substances, or the like. Suitablecarriers are typically large, slowly metabolized molecules such asproteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, lipid aggregates, or thelike.

For the preparation of vaccines, the recombinant poxvirus according tothe invention is converted into a physiologically acceptable form. Thiscan be done based on the experience in the preparation of poxvirusvaccines used for vaccination against smallpox (as described by Stickl,H. et al. 1974 Dtsch Med Wochenschr. 99:2386-2392). For example, thepurified virus is stored at −80° C. with a titer of 5×10E8 TCID₅₀/mlformulated in about 10 mM Tris, 140 mM NaCl pH 7.4. For the preparationof vaccine shots, e.g., 10E2-10E8 particles of the virus are lyophilizedin 100 ml of phosphate-buffered saline (PBS) in the presence of 2%peptone and 1% human albumin in an ampoule, preferably a glass ampoule.Alternatively, the vaccine shots can be produced by stepwisefreeze-drying of the virus in a formulation. This formulation cancontain additional additives such as mannitol, dextran, sugar, glycine,lactose or polyvinylpyrrolidone or other aids such as antioxidants orinert gas, stabilizers or recombinant proteins (e.g., human serumalbumin) suitable for in vivo administration. The glass ampoule is thensealed and can be stored between 4° C. and room temperature for severalmonths. However, as long as no need exists the ampoule is storedpreferably at temperatures below −20° C.

For vaccination or therapy the lyophilisate can be dissolved in 0.1 to0.5 ml of an aqueous solution, preferably physiological saline or Trisbuffer, and administered either systemically or locally, i.e.,parenterally, subcutaneous, intramuscularly, by scarification or anyother path of administration know to the skilled practitioner. The modeof administration, the dose and the number of administrations can beoptimized by those skilled in the art in a known manner. However, mostcommonly a patient is vaccinated with a second shot about one month tosix weeks after the first vaccination shot.

One embodiment of the present invention is a method to generate animmune response against an antigen. Such a response can be a CD8⁺ T cellimmune response or an antibody response. More particularly, the presentinvention relates to “prime and boost” immunization regimes in which theimmune response induced by administration of a priming composition isboosted by administration of a boosting composition. The presentinvention is based on prior experimental demonstration that effectiveboosting can be achieved using modified vaccinia Ankara (MVA) vectors,following priming with any of a variety of different types of primingcompositions including recombinant MVA itself.

A major protective component of the immune response against a number ofpathogens is mediated by T lymphocytes of the CD8⁺ type, also known ascytotoxic T lymphocytes (CTL). An important function of CD8⁺ cells issecretion of gamma interferon (IFNγ), and this provides a measure ofCD8⁺ T cell immune response. A second component of the immune responseis antibody directed to the proteins of the pathogen.

The present invention employs MVA which, as prior experiments show, hasbeen found to be an effective means for providing a boost to a CD8⁺ Tcell immune response primed to antigen using any of a variety ofdifferent priming compositions and also eliciting an antibody response.

Notably, prior experimental work demonstrates that use of predecessorsof the present invention allows for recombinant MVA virus expressing anHIV antigen to boost a CD8⁺ T cell immune response primed by a DNAvaccine and also eliciting an antibody response. The MVA may be found toinduce a CD8⁺ T cell response after immunization. Recombinant MVA mayalso be shown to prime an immune response that is boosted by one or moreinoculations of recombinant MVA.

Non-human primates immunized with plasmid DNA and boosted with the MVAwere effectively protected against intramucosal challenge with livevirus (Amara et al 2001 Science 292:69-74). Advantageously, theinventors contemplate that a vaccination regime using intradermal,intramuscular or mucosal immunization for both prime and boost can beemployed, constituting a general immunization regime suitable forinducing CD8⁺ T cells and also eliciting an antibody response, e.g., inhumans.

The present invention in various aspects and embodiments employs an MVAvector encoding an HIV antigen for boosting a CD8⁺ T cell immuneresponse to the antigen primed by previous administration of nucleicacid encoding the antigen and also eliciting an antibody response.

A general aspect of the present invention provides for the use of an MVAvector for boosting a CD8⁺ T cell immune response to an HIV antigen andalso eliciting an antibody response.

One aspect of the present invention provides a method of boosting a CD8⁺T cell immune response to an HIV antigen in an individual, and alsoeliciting an antibody response, the method including provision in theindividual of an MVA vector including nucleic acid encoding the antigenoperably linked to regulatory sequences for production of antigen in theindividual by expression from the nucleic acid, whereby a CD8⁺ T cellimmune response to the antigen previously primed in the individual isboosted.

An immune response to an HIV antigen may be primed by immunization withplasmid DNA or by infection with an infectious agent.

A further aspect of the invention provides a method of inducing a CD8⁺ Tcell immune response to an HIV antigen in an individual, and alsoeliciting an antibody response, the method comprising administering tothe individual a priming composition comprising nucleic acid encodingthe antigen and then administering a boosting composition whichcomprises an MVA vector including nucleic acid encoding the antigenoperably linked to regulatory sequences for production of antigen in theindividual by expression from the nucleic acid.

A further aspect provides for use of an MVA vector, as disclosed, in themanufacture of a medicament for administration to a mammal to boost aCD8⁺ T cell immune response to an HIV antigen, and also eliciting anantibody response. Such a medicament is generally for administrationfollowing prior administration of a priming composition comprisingnucleic acid encoding the antigen.

The priming composition may comprise DNA encoding the antigen, such DNApreferably being in the form of a circular plasmid that is not capableof replicating in mammalian cells. Any selectable marker should not beresistance to an antibiotic used clinically, so for example Kanamycinresistance is preferred to Ampicillin resistance. Antigen expressionshould be driven by a promoter which is active in mammalian cells, forinstance the cytomegalovirus immediate early (CMV IE) promoter.

In particular embodiments of the various aspects of the presentinvention, administration of a priming composition is followed byboosting with a boosting composition, or first and second boostingcompositions, the first and second boosting compositions being the sameor different from one another. Still further boosting compositions maybe employed without departing from the present invention. In oneembodiment, a triple immunization regime employs DNA, then adenovirus asa first boosting composition, then MVA as a second boosting composition,optionally followed by a further (third) boosting composition orsubsequent boosting administration of one or other or both of the sameor different vectors. Another option is DNA then MVA then adenovirus,optionally followed by subsequent boosting administration of one orother or both of the same or different vectors.

The antigen to be encoded in respective priming and boostingcompositions (however many boosting compositions are employed) need notbe identical, but should share at least one CD8⁺ T cell epitope. Theantigen may correspond to a complete antigen, or a fragment thereof.Peptide epitopes or artificial strings of epitopes may be employed, moreefficiently cutting out unnecessary protein sequence in the antigen andencoding sequence in the vector or vectors. One or more additionalepitopes may be included, for instance epitopes which are recognized byT helper cells, especially epitopes recognized in individuals ofdifferent HLA types.

An HIV antigen of the invention to be encoded by a recombinant MVA virusincludes polypeptides having immunogenic activity elicited by an aminoacid sequence of an HIV Env, Gag, Pol, Vif, Vpr, Tat, Rev, Vpu, or Nefamino acid sequence as at least one CD8⁺ T cell epitope. This amino acidsequence substantially corresponds to at least one 10-900 amino acidfragment and/or consensus sequence of a known HIV Env or Pol; or atleast one 10-450 amino acid fragment and/or consensus sequence of aknown HIV Gag; or at least one 10-100 amino acid fragment and/orconsensus sequence of a known HIV Vif, Vpr, Tat, Rev, Vpu, or Nef.

Although a full length Env precursor sequence is presented for use inthe present invention, Env is optionally deleted of subsequences. Forexample, regions of the gp120 surface and gp41 transmembrane cleavageproducts can be deleted.

Although a full length Gag precursor sequence is presented for use inthe present invention, Gag is optionally deleted of subsequences. Forexample, regions of the matrix protein (p17), regions of the capsidprotein (p24), regions of the nucleocapsid protein (p7), and regions ofp6 (the C-terminal peptide of the Gag polyprotein) can be deleted.

Although a full length Pol precursor sequence is presented for use inthe present invention, Pol is optionally deleted of subsequences. Forexample, regions of the protease protein (p10), regions of the reversetranscriptase protein (p66/p51), and regions of the integrase protein(p32) can be deleted.

Such an HIV Env, Gag, or Pol can have overall identity of at least 50%to a known Env, Gag, or Pol protein amino acid sequence, such as 50-99%identity, or any range or value therein, while eliciting an immunogenicresponse against at least one strain of an HIV.

Percent identity can be determined, for example, by comparing sequenceinformation using the GAP computer program, version 6.0, available fromthe University of Wisconsin Genetics Computer Group (UWGCG). The GAPprogram utilizes the alignment method of Needleman and Wunsch (J MolBiol 1970 48:443), as revised by Smith and Waterman (Adv Appl Math 19812:482). Briefly, the GAP program defines identity as the number ofaligned symbols (i.e., nucleotides or amino acids) which are identical,divided by the total number of symbols in the shorter of the twosequences. The preferred default parameters for the GAP program include:(1) a unitary comparison matrix (containing a value of 1 for identitiesand 0 for non-identities) and the weighted comparison matrix of Gribskovand Burgess (Nucl Acids Res 1986 14:6745), as described by Schwartz andDayhoff (eds., Atlas of Protein Sequence and Structure, NationalBiomedical Research Foundation, Washington, D.C. 1979, pp. 353-358); (2)a penalty of 3.0 for each gap and an additional 0.10 penalty for eachsymbol in each gap; and (3) no penalty for end gaps.

In a preferred embodiment, an Env of the present invention is a variantform of at least one HIV envelope protein. Preferably, the Env iscomposed of gp120 and the membrane-spanning and ectodomain of gp41 butlacks part or all of the cytoplasmic domain of gp41.

Known HIV sequences are readily available from commercial andinstitutional HIV sequence databases, such as GENBANK, or as publishedcompilations, such as Myers et al. eds., Human Retroviruses and AIDS, ACompilation and Analysis of Nucleic Acid and Amino Acid Sequences, Vol.I and II, Theoretical Biology and Biophysics, Los Alamos, N. Mex.(1993), or on the world wide web at hiv-web.lanl.gov/.

Substitutions or insertions of an HIV Env, Gag, or Pol to obtain anadditional HIV Env, Gag, or Pol, encoded by a nucleic acid for use in arecombinant MVA virus of the present invention, can includesubstitutions or insertions of at least one amino acid residue (e.g.,1-25 amino acids). Alternatively, at least one amino acid (e.g., 1-25amino acids) can be deleted from an HIV Env, Gag, or Pol sequence.Preferably, such substitutions, insertions or deletions are identifiedbased on safety features, expression levels, immunogenicity andcompatibility with high replication rates of MVA.

Amino acid sequence variations in an HIV Env, Gag, or Pol of the presentinvention can be prepared e.g., by mutations in the DNA. Such HIV Env,Gag, or Pol include, for example, deletions, insertions or substitutionsof nucleotides coding for different amino acid residues within the aminoacid sequence. Obviously, mutations that will be made in nucleic acidencoding an HIV Env, Gag, or Pol must not place the sequence out ofreading frame and preferably will not create complementary domains thatcould produce secondary mRNA structures.

HIV Env, Gag, or Pol-encoding nucleic acid of the present invention canalso be prepared by amplification or site-directed mutagenesis ofnucleotides in DNA or RNA encoding an HIV Env, Gag, or Pol andthereafter synthesizing or reverse transcribing the encoding DNA toproduce DNA or RNA encoding an HIV Env, Gag, or Pol, based on theteaching and guidance presented herein.

Recombinant MVA viruses expressing HIV Env, Gag, or Pol of the presentinvention, include a finite set of HIV Env, Gag, or Pol-encodingsequences as substitution nucleotides that can be routinely obtained byone of ordinary skill in the art, without undue experimentation, basedon the teachings and guidance presented herein. For a detaileddescription of protein chemistry and structure, see Schulz, G. E. etal., 1978 Principles of Protein Structure, Springer-Verlag, New York,N.Y., and Creighton, T. E., 1983 Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, Calif. For apresentation of nucleotide sequence substitutions, such as codonpreferences, see Ausubel et al. eds. Current Protocols in MolecularBiology, Greene Publishing Assoc., New York, N.Y. 1994 at§§A.1.1-A.1.24, and Sambrook, J. et al. 1989 Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. at Appendices C and D.

Thus, one of ordinary skill in the art, given the teachings and guidancepresented herein, will know how to substitute other amino acid residuesin other positions of an HIV env, gag, or pol DNA or RNA to obtainalternative HIV Env, Gag, or Pol, including substitutional, deletionalor insertional variants.

Within the MVA vector, regulatory sequences for expression of theencoded antigen will include a promoter. By “promoter” is meant asequence of nucleotides from which transcription may be initiated of DNAoperably linked downstream (i.e., in the 3′ direction on the sensestrand of double-stranded DNA). “Operably linked” means joined as partof the same nucleic acid molecule, suitably positioned and oriented fortranscription to be initiated from the promoter. DNA operably linked toa promoter is “under transcriptional initiation regulation” of thepromoter. Other regulatory sequences including terminator fragments,polyadenylation sequences, marker genes and other sequences may beincluded as appropriate, in accordance with the knowledge and practiceof the ordinary person skilled in the art: see, for example, Moss, B.(2001). Poxviridae: the viruses and their replication. In FieldsVirology, D. M. Knipe, and P. M. Howley, eds. (Philadelphia, LippincottWilliams & Wilkins), pp. 2849-2883. Many known techniques and protocolsfor manipulation of nucleic acid, for example in preparation of nucleicacid constructs, mutagenesis, sequencing, introduction of DNA into cellsand gene expression, and analysis of proteins, are described in detailin Current Protocols in Molecular Biology, 1998 Ausubel et al. eds.,John Wiley & Sons.

Promoters for use in aspects and embodiments of the present inventionmay be compatible with poxvirus expression systems and include natural,modified and synthetic sequences.

Either or both of the priming and boosting compositions may include anadjuvant, such as granulocyte macrophage-colony stimulating factor(GM-CSF) or encoding nucleic acid therefor.

Administration of the boosting composition is generally about 1 to 6months after administration of the priming composition, preferably about1 to 3 months.

Preferably, administration of priming composition, boosting composition,or both priming and boosting compositions, is intradermal, intramuscularor mucosal immunization.

Administration of MVA vaccines may be achieved by using a needle toinject a suspension of the virus. An alternative is the use of aneedleless injection device to administer a virus suspension (using,e.g., Biojector™ needleless injector) or a resuspended freeze-driedpowder containing the vaccine, providing for manufacturing individuallyprepared doses that do not need cold storage. This would be a greatadvantage for a vaccine that is needed in rural areas of Africa.

MVA is a virus with an excellent safety record in human immunizations.The generation of recombinant viruses can be accomplished simply, andthey can be manufactured reproducibly in large quantities. Intradermal,intramuscular or mucosal administration of recombinant MVA virus istherefore highly suitable for prophylactic or therapeutic vaccination ofhumans against AIDS which can be controlled by a CD8⁺ T cell response.

The individual may have AIDS such that delivery of the antigen andgeneration of a CD8⁺ T cell immune response to the antigen is of benefitor has a therapeutically beneficial effect.

Most likely, administration will have prophylactic aim to generate animmune response against HIV or AIDS before infection or development ofsymptoms.

Components to be administered in accordance with the present inventionmay be formulated in pharmaceutical compositions. These compositions maycomprise a pharmaceutically acceptable excipient, carrier, buffer,stabilizer or other materials well known to those skilled in the art.Such materials should be non-toxic and should not interfere with theefficacy of the active ingredient. The precise nature of the carrier orother material may depend on the route of administration, e.g.,intravenous, cutaneous or subcutaneous, nasal, intramuscular,intraperitoneal routes.

As noted, administration is preferably intradermal, intramuscular ormucosal.

Physiological saline solution, dextrose or other saccharide solution orglycols such as ethylene glycol, propylene glycol or polyethylene glycolmay be included.

For intravenous, cutaneous, subcutaneous, intramuscular or mucosalinjection, or injection at the site of affliction, the active ingredientwill be in the form of a parenterally acceptable aqueous solution whichis pyrogen-free and has suitable pH, isotonicity and stability. Those ofrelevant skill in the art are well able to prepare suitable solutionsusing, for example, isotonic vehicles such as Sodium Chloride Injection,Ringer's Injection, Lactated Ringer's Injection. Preservatives,stabilizers, buffers, antioxidants and/or other additives may beincluded as required.

A slow-release formulation may be employed.

Following production of MVA particles and optional formulation of suchparticles into compositions, the particles may be administered to anindividual, particularly human or other primate. Administration may beto another mammal, e.g., rodent such as mouse, rat or hamster, guineapig, rabbit, sheep, goat, pig, horse, cow, donkey, dog or cat.

Administration is preferably in a “prophylactically effective amount” ora “therapeutically effective amount” (as the case may be, althoughprophylaxis may be considered therapy), this being sufficient to showbenefit to the individual. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g., decisions ondosage etc, is within the responsibility of general practitioners andother medical doctors, or in a veterinary context a veterinarian, andtypically takes account of the disorder to be treated, the condition ofthe individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found in Remington'sPharmaceutical Sciences, 16th edition, 1980, Osol, A. (ed.).

In one preferred regimen, DNA is administered at a dose of 300 μg to 3mg/injection, followed by MVA at a dose of 10⁶ to 10⁹ infectious virusparticles/injection.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

Delivery to a non-human mammal need not be for a therapeutic purpose,but may be for use in an experimental context, for instance ininvestigation of mechanisms of immune responses to an antigen ofinterest, e.g., protection against HIV or AIDS.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the embodiments, and are not intended to limit the scope ofwhat the inventors regard as their invention nor are they intended torepresent that the experiments below are all or the only experimentsperformed. Efforts have been made to ensure accuracy with respect tonumbers used (e.g. amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is weight averagemolecular weight, and temperature is in degrees Celsius. Standardabbreviations are used.

Example 1

The following Example demonstrates a shuttle plasmid, recombinantMVA/HIV1 clinical vaccine construct and mechanism for retention ofintact foreign gene inserts in recombinant MVA by codon alteration ofthe foreign gene and insertion of the foreign gene between two vacciniavirus essential genes. The disclosure provides mechanisms for:

-   -   retention of intact foreign genes by inserting them between two        vaccinia virus genes that are essential for MVA replication.        Deletion of the foreign gene can provide a significant growth        advantage for the recombinant MVA allowing it to compete with        MVA containing the intact foreign gene upon repeated passage.        However, most deletions of a foreign gene include loss of some        part of the flanking vaccinia virus DNA. If that vaccinia virus        DNA is essential, then those viruses with deletions will not        replicate and compete with the MVA containing the intact foreign        gene. This methodology will be useful in production of        recombinant vaccinia viruses that must be amplified to large        scale such as for use in clinical trials, and    -   stabilizing foreign gene inserts by alteration of specific “hot        spots” that otherwise readily undergo mutation after repeated        passage of the recombinant virus. This methodology is useful in        production of recombinant viruses that must be amplified to        large scale such as for use in clinical trials.

And describes:

-   -   the shuttle plasmid, pLW-73, used for insertion of a foreign        gene between 2 essential vaccinia virus genes; and    -   the recombinant MVA/HIV-1 clinical vaccine construct MVA/UGD4d,        a material that embodies use of these two mechanisms.        Generation of Stable Recombinant MVA Viruses        Modified vaccinia virus Ankara (MVA) recombinants expressing env        and gagpol genes from HIV-1 isolates from different geographical        locations were constructed. The foreign genes were inserted into        2 sites, Deletion II and Deletion III of MVA. The stability of        these genes after repeated passage of recombinant MVA in tissue        culture has proven to be variable. The inventors demonstrated        that the instability was due to either deletion of the entire        foreign gene and some flanking DNA or specific point mutations        resulting in propagation of progeny virions that have a growth        advantage because they do not express the foreign gene. Here the        inventors describe two novel methods of retaining the intact        foreign gene recombinant MVA. First, the inventors constructed a        transfer vector that directs insertion of a foreign gene between        two essential vaccinia virus genes in the conserved central        region of the genome. Use of this site for insertion of genes        prevents the outgrowth of variants containing large deletions        that include the essential vaccinia virus DNA. In addition, this        plasmid can be used for insertion of additional genes into        recombinant viruses. Second, analysis of isolates with point        mutations revealed certain “hot spots” with a propensity for        insertion or deletion of a single base that causes premature        termination during translation. The inventors showed that        generation of silent mutations in these sites resulted in        stabilization of the inserted gene.

I. Novel Transfer Vector Construction and Application

Construction of Novel Transfer Vector, pLW-73

1. The central region of the MVA genome, K7R-A24R, was examined for 1)pairs of genes conserved in the poxvirus family or chordopoxvirussubfamily and 2) genes that are in opposite orientation such that their3′ ends are in close proximity, thereby providing an insertion site thatwould not disrupt a vaccina promoter. The site chosen as the newinsertion site was between two essential genes, I8R and G1L.

2. The left flank of the new vector was constructed in the followingway: Plasmid LAS-1 was cut with restriction enzymes EcoRI and XhoI toremove the del III MVA flank, GFP, and direct repeat of MVA flank. Thisinsert was cut with AscI and SacI and the GFP fragment was isolated.Five hundred thirty one base pairs at the end of the I8R gene (includingthe TAA stop codon) was PCR amplified with EcoRI and AscI restrictionsites on the ends of the PCR product. PCR amplification of 229 basepairs of the direct repeat (from the end of the 18R gene including theTAA stop codon) was performed with oligonucleotides containing SacI andXhoI restriction sites. All four pieces of DNA, 1) the vector backbonewith EcoRI and Xho I ends, 2) new left flank containing end of I8R withEcoRI and AscI ends, 3) GFP with AcsI and SacI ends and the 4) directrepeat of the I8R flank with SacI and XhoI ends were ligated together tomake plasmid pLW-72.

3. The right flank was made as follows: pLW-72 was cut with restrictionenzymes PstI and HindIII to release del III flank of the MVA in theplasmid. Seven hundred and two base pairs at the end of the G1L gene wasPCR amplified with PstI and HindIII restriction enzyme sites on the endsand ligated into the pLW-72 vector to make pLW-73 (FIG. 7). The sequenceof pLW-73 is given in FIG. 8.

4. The salient features of pLW-73 are: 1) the vector was designed forinsertion of foreign genes between essential genes in MVA genome. Theleft flank consists of end of I8R gene and right flank consists of endof G1L gene. 2) the GFP gene is included for easy initial selection ofrecombinant virus 3) the GFP is flanked by direct repeats of the I8Rgene which allows for transient expression of GFP as the GFP will belost upon repeated passage of the recombinant virus. Referring to WO2004/087201, features 2 and 3 were also contained in earlier plasmidsused for making MVA/HIV recombinants, pLAS-1 and pLAS-2.

Application of pLW-73

1. The env gene from the clade B ADA isolate of HIV-1 was cloned intopLW-73 and a recombinant MVA virus was made. DNA sequencing confirmedthe location and integrity of the env gene.

2. A recombinant MVA virus expressing the Ugandan clade D (isolateAO7412) env gene (FIG. 9) in the Deletion II site of MVA proved to beunstable, i.e., after repeated serial passage in culture, the gene wasdeleted from a significant portion of the virus progeny. The same genewas then cloned into pLW-73 and a recombinant MVA virus was made andcharacterized. The env gene insert was stable after repeated serialpassage (8×) in culture i.e., no deletions of the inserted gene or theMVA flanking region were found. In addition, no other mutations arosewhen the gene was inserted into this site.

II. Point Mutation of “Hot Spots”

Analysis of Point Mutations

A recombinant MVA virus expressing the Ugandan Clade D (isolate AO3349)gagpol gene in the Deletion III site of MVA proved to be unstable. Themajor genetic alteration was the generation of single point mutations inruns of 4-6 G or C residues (Table 3). In addition, similar pointmutations were found in non-staining plaques from similar recombinantviruses expressing the gagpol genes from a Kenyan clade A isolate and aTanzanian clade C isolate of HIV-1.

Mutagenesis of Hot Spots and Analysis of Stability in Recombinant Virus

Using site-directed mutagenesis, silent mutations were made in 6 suchregions of the gag gene from the Ugandan HIV-1 isolate. This alteredgene, UGD 4d gagpol orf (FIG. 10), was cloned into pLAS-1 and recombinedinto the same Deletion III site of MVA as was done in construction ofthe unstable virus. After repeated serial passage (8×) in culture, nonon-expressing plaques were found. DNA sequencing of the passage 8 virusstock verified that the integrity of the gagpol gene was maintained.

III. Double Recombinant Construction

MVA/UGD4d Virus

MVA/UGD4d virus, a recombinant virus that expresses the Ugandan subtypeD AO7412 envelope and the AO3349 gagpol, was constructed in thefollowing way: The envelope and gagpol genes were inserted into MVA1974/NIH Clone 1 by homologous recombination utilizing shuttle plasmidspLW-73 and pLAS-1, respectively. MVA/UGD4d was isolated by 6 rounds ofplaque purification in chicken embryo fibroblast cells and subsequentlyamplified and characterized.

Summary

1. A plasmid transfer vector was constructed that directs recombinationof a foreign gene between two essential genes, I8R and G1L, in theconserved central region of the MVA genome. The use of this site wasshown to inhibit selection of mutant viruses with deletions of insertedgene/MVA flanks.

2. Highly mutable runs of G and C residues were altered by site-directedmutagenesis and silent mutations in the coding sequence were generated.This change was shown to stabilize the gene when inserted into DeletionIII of MVA.

3. Utilizing these two methods above, UGD4d double MVA recombinant thatstably expresses both the env and gagpol of Ugandan Clade D wasconstructed.

Example 2

Recombinant MVAs expressing HIV-1 env and gagpol genes from manydifferent isolates have been made. The stability of inserted genes afterrepeated passage in tissue culture has proven to be variable. Here theinventors (1) demonstrate that the instability represents a combinationof spontaneous mutation or deletion of the inserted gene and selectionfor non-expressing mutants and (2) describe novel methods for reducinginstability.

Overview

Recombinant MVAs expressing env and gagpol from many different isolateswere constructed. Each virus was subjected to repeated passages inchicken embryo fibroblast cells to mimic the large-scale amplificationrequired for production of virus for clinical trials. Insert stabilitywas monitored by env and gag immunostaining of individual plaques. Forsome recombinant viruses, env and/or gag expression was found to berapidly lost in a significant fraction of the virus population. Toidentify the mechanism(s) of loss of expression, individual plaques wereisolated and the nature of the mutations was characterized. In somecases, specific DNA sequences with propensity to mutate by addition ordeletion of a single nucleotide were identified. Generation of suchmutations could be avoided by altering codons without changing thepredicted translation product. In other cases, loss of expression wascaused by large deletions that frequently extended into flankingnon-essential MVA genes. To prevent this from occurring, a new shuttleplasmid was constructed that was designed to direct insertion of foreigngenes between two essential MVA genes. Recombination into this sitereduced deletions of the foreign DNA. In one case, however, the toxicityassociated with high-level HIV env expression was so severe that theselection of rare mutants still resulted in an unstable population. Inthis case, only truncation of the transmembrane domain of env allowedthe construction of a stable recombinant MVA.

Generation of Recombinant MVAs and Analysis of Stability of InsertedGenes

Env and gagpol genes were cloned into MVA shuttle vectors. Expressionand function were analyzed by transient expression assays. Gagpol wasrecombined into MVA 1974/NIH Clone 1. Recombinant MVA were plaquepurified with 6-8 rounds followed by amplification of virus. Env wasrecombined into the MVA/gagpol isolate and double-recombinant MVA (FIG.11A) were plaque purified with 6-8 rounds and were amplified. To assessthe stability of inserts, virus was serially passaged in CEF cells usinga multiplicity of infection (m.o.i.) of ˜1 pfu/cell to mimic large-scaleproduction. Stability was evaluated by determining the percentage ofcells expressing env or gag, as determined by immunostaining withmonoclonal antibodies (FIG. 11B).

Stability of Recombinant MVAs

Recombinant MVAs expressing genes from HIV-1 isolates from differentgeographical locations were constructed. The env and gagpol genes wereinserted into deletions II and III of MVA, respectively; both undercontrol of the modified H5 promoter. The stability of env and gagpolgenes from seven recombinant MVAs is shown in Table 4. Varying degreesof instability were observed in the seven viruses. In MVA/65A/G,expression of env was rapidly lost with only 25% of virions expressingenv by passage 6. In MVA/UGD4a, both env and gagpol expression wereincreasingly lost with successive virus passages. Since at least 6-7passages are required for production of a lot of virus for a Phase Itrial, these two viruses were deemed unsuitable.

Analysis of Expression of MVA/65A/G

Referring to FIG. 12, thirteen plaques were randomly picked from P3 andP5 of MVA/65A/G and analyzed by immunostaining with T-24 mAb (bindingsite shown on a), Western blotting, PCR, and sequencing. Five types ofplaques were found and the number of these plaques obtained for eachtype are given at right of FIG. 12. Plaques a, b, and c stained, but band c were truncated versions due to base substitution (causing stopcodon) (b) and deletion of the end of the env gene and part of MVA flank(c). Nonstaining plaques d and e resulted from addition of G to a 5G runcausing a frameshift (d) and large deletion of entire env gene and partsof MVA flanks (e). Thus, base pair addition, substitution, and deletionsall contributed to unstable expression of the env gene in MVA/65A/G.This A/G env, the most unstable example worked with, was picked to studymodifications that might enhance stability.

Modifications to A/G Constructs to Increase Stability

1. Synthetic envelope was made by removing 4 and 5 G and C runs bysilent mutations to prevent point mutations.

2. Vector I8/G1, i.e., pLW-73. was constructed with an insertion sitebetween essential genes I8R and G1L to prevent deletions of genes andMVA flanks from being viable. The ends of the I8R (500 bp) and G1L (750bp) genes of MVA were amplified by PCR and inserted into a vectorcontaining vaccinia virus early/late mH5 promoter controlling foreigngene expression. This I8/G1 vector was used to insert foreign genes intoMVA by homologous recombination (FIG. 13). Deletions of inserted genesand MVA flanking the inserted gene would not be viable because parts ofessential genes would be deleted. Therefore, viruses with thesemutations would not be able to overgrow the population with their normalgrowth advantage.

3. A/G gp140 envelope was mutated by deleting the transmembrane domainand the cytoplasmic tail of gp41, resulting in a secreted protein.

Testing Modifications to Increase Stability

Seven single recombinant viruses were made with env modifications and/oruse of new vector as shown in FIG. 14. Five plaques of each virus wereisolated and passaged independently in CEF to determine if modificationsenhanced envelope stable expression. Passaged plaques were analyzed byimmunostaining with mAb T-43 (binding site mapped to 101-125aa of env),Western blotting, PCR, and sequencing.

Env Expression after Plaque Passages

Referring to FIG. 15, five independently passaged plaque isolates ofeach of the 7 recombinants listed above, were characterized at passages1, 3, 5, and 7 by immunostaining with mAb T-43 (binds between101-125a.a. in gp120). Four of 7 viruses (FIG. 15, a, b, c, e) hadunstable protein expression in each of the 5 passaged plaques; twoplaque passages of (FIG. 15 f) also had unstable env expression. Theseincluded viruses with the synthetic env in both del II (FIG. 15 c) andin the essential gene site (FIG. 15 f) of MVA genome. Only recombinantviruses containing the envelope as truncated, secreted gp140 remainedstably expressing envelope (FIGS. 15, d and g).

Western Blotting, PCR and Sequence Analyses

From selected plaque passages, clones were picked to analyze proteinexpression by Western blotting, PCR, and sequence analysis (FIG. 16).For Western blot analysis, T-24 and T-32 binding at the beginning andend of the clade A envelope, respectively, were used in order todetermine if only partial or full length envelope was being made.Control viruses, marked c, are at the right of each blot. For the threeviruses made in deletion II of MVA (FIGS. 16 a, b, and c), only in FIG.16 c (i.e., gp140 clones), were all the clones expressing detectableprotein in Western. This protein (as measured by T-32) was nottruncated. When envelope was inserted into the essential gene site byvector I8/G1 (FIGS. 16 d, e and f), again, only the gp140 envelope wasbeing expressed in all clones and was not truncated. Although use ofI8/G1 vector did not prevent mutations to the env sequence, it didprevent deletions which had been seen in envelope inserted into del II.(Note positive PCR products from all clones tested from I8/G1 vector,but negative PCR products from clones tested using del II vector.)

Expression of Env in Clade A/G Double Recombinant

Based on previous results with single env analysis, double recombinantsexpressing gagpol with either gp140 or the synthetic gp160 gene weremade and tested for stability of env expression (FIG. 17). Five plaqueswere isolated from each as previously described, and passaged 7 times toanalyze stability of env expression. At passage 7, the passaged plaqueswere immunostained with both T-43 and T-32 mAbs (which bind to gp120 andgp41, respectively). With T-43 mAb, one of five clones of recombinantexpressing synthetic envelope consisted of only non-staining plaques.Subsequent T-32 staining of these plaques showed another plaque hadtruncated envelope expression. All passaged plaques from doublerecombinant containing gp140 envelope appeared stable by both T-43 andT-32 immunostaining. Titers were also 2 logs higher than with the otherdouble recombinant. Thus a clade A/G double recombinant stablyexpressing envelope could only be made with gp140 envelope.

Recombinant Viruses Expressing Env and Gagpol from Ugandan HIV-1Isolates

Recombinant MVA viruses expressing HIV-1 env and gagpol genes fromUgandan isolates AO7412 and AO3349 were constructed as shown in FIG. 18.Four to six independent isolates of each were serially passaged and bothgenes were found to be unstable whether expressed alone or incombination (Table 5). In contrast, expression of gp140 instead ofmembrane bound gp160 resulted in stability of the env gene after serialpassage (FIG. 18 and Table 5).

MVA/UGD4a—Analysis of Non-Staining Env Plaques

To determine the mechanism of instability, 24 individual non-stainingplaques (using Mab T-43) were isolated from passage 6 of MVA/UGD4a,amplified, and characterized. Two small deletions (1.2 and 0.3 kb) wereidentified by PCR amplification and DNA sequencing (FIG. 19). All otherisolates contained very large deletions that extended into the flankingMVA. The approximate break-points for these deletions were identifiedusing primer pairs from within the env gene or flanking MVA regions.

Modification of UGD Env Gene in Recombinant MVA

To ameliorate the problem of instability of the UGD env gene, the AO7412env gene was inserted into MVA using the new vector, I8/G1, whichdirects recombination of a foreign gene between 2 essential vacciniavirus genes, I8 and G1 and uses the modified H5 promoter (FIG. 20). Fourindependent plaques were serially passaged and analyzed for envexpression by immunostaining with Mabs T-43 and T-32 at passage 5. Inall isolates, the gene was stable (Table 6).

MVA/UGD4b—Analysis of Non-Staining Gag Plaques

To determine the mechanism of instability of the gag gene, 8 individualnon-staining plaques (using Mab 183-H12-5C—NIAID AIDS Repository) werepicked from passage 6 of MVA/UGD4b, amplified, and the gagpol insert wassequenced (Table 7). In 7 isolates, an insertion or deletion of a singleG residue at position 564-569 was found. In one isolate, a C residue wasdeleted from the sequence CCCC at position 530-534. Furthermore,non-staining plaques from high-passage stocks of MVA/KEA and MVA/TZCrevealed a similar hot-spot for mutation, i.e., position 564-569.Examination of the full sequence of the UGD AO7412 gagpol genedemonstrated 22 runs of 4 or more G or C residues (FIG. 21).

Modification of UGD Gagpol Gene in Recombinant MVA

Since the mechanism of instability of the gagpol gene was primarilyinsertion or deletion of a single nucleotide within a run of 4-6 G or Cresidues, the strategy to improve the stability of this gene was togenerate silent mutations at such sites. Thus, site-directed mutagenesisat 6 sites in p17 and p24 gag (Table 3) was employed. The resultingcodon altered (c.a.) gene inserted into MVA at the same location, i.e.,Deletion III, proved to be stable upon serial passage (FIG. 22 and Table8).

Construction of Stable, Recombinant MVA Expressing UGD Env and Gagpol

A recombinant virus expressing the UGD env gene in the I8/G1 locus andthe codon altered gagpol gene in Deletion III of MVA was constructed(FIG. 23). Serial passage demonstrated no instability of either gene.Furthermore, the level of protein expression and DNA sequence wereunaltered during passage (Table 9).

Conclusions

Instability of env and gagpol inserts is attributed to the generation ofpoint mutations and deletions and the growth advantage of non-expressingMVA mutants. Instability can generally be reduced by codon alterationand/or insertion into an essential region of the MVA genome (MVA/UGD4d)but env had to be altered in one case (MVA/65A/G).

Example 3 Immunogenicity of MVA/UGD4d in BALB/c Mice

Groups of 10 mice each were immunized by the intraperitoneal route witheither 10⁶ or 10⁷ infectious units of MVA/UGD4d. Groups of 5 mice eachwere similarly immunized with parental MVA-1974. Mice were immunized atweeks 0 and 3 and bled at weeks 0, 3, and 5. Spleens were harvested atweek 5.

Cellular responses were measured in fresh splenocytes by intracellularcytokine staining. Splenocytes were separately stimulated with thefollowing: 1) immunodominant gag peptide (AMQMLKETI (SEQ ID NO: 6)), 2)env peptides (DTEVHNVWATHACVP (SEQ ID NO: 7) and QQQSNLLRAIEAQQH (SEQ IDNO: 8)), 3) pol peptides (8 peptides with single amino acid variants ofELRQHLLRWGLTT (SEQ ID NO: 9) and HGVYYDPSKDLIAE (SEQ ID NO: 10)), and 4)MVA.

Cells were stained for surface expression of CD4 and CD8 and then forintracellular expression of IFN-γ and either IL2 or TNF. As shown inFIG. 24, MVA/UGD4d elicited CD8/IFN-γ responses to the gag peptide, polpeptides, and MVA. The gag peptide responses were multifunctional,expressing both IFN-γ and either IL2 or TNF. Also, CD4/IFN-γ responseswere elicited to the pool of env peptides.

Humoral responses were measured by ELISA (FIG. 25). Strong responses toUGD env were demonstrated at 3 weeks after one immunization and wereboosted by the second immunization. In addition, strong vaccinia virusresponses were elicited after one and two immunizations.

TABLE 3 MVA/UGD Nucleotide Changes Made toEliminate Runs of G and C (HIV-1 isolate AO3349) Nucleotide # startingOriginal Modified with ATG Sequence Sequence 28-32 GGGGG GGAGG 70-74GGGGG GGAGG 408-411 GGGG GGGA 530-533 CCCC CACC 564-569 GGGGGG AGGAGG686-689 GGGG GAGG

TABLE 4 Stability of Recombinant MVAs Percent non-staining plaquespassage passage passage passage Geographic LVD seed 3/4 6/7 8/9 10-13vaccine lot Virus Clade origin env gag env gag env gag env gag env gagenv gag KEA5b A Kenya <1 <1 0.13 0.33 0.34 0.36 0.54 2.4 0.64 0.77 65A/GA/G Ivory <2 <1 28 1 75 Coast 62B B US <1 <1 <1 <1 6 <1 10 1 TZCa CTanzania <1 <1 <1 <1 1.7 2.8 3.6 3.7 71C C India <1 <1 <1 1 <1 2 12 14UGD4a D Uganda <1 <1 3 0.28 6.7 6 12.2 17.4 CMDR E/A Thailand <1 <1 <1<1 <1 <1 <1 <1

TABLE 5 Recombinant Viruses Expressing env and gagpol from Ugandan HIV-1isolates % non-staining passage env gag UGD4a 9 12.2 17.4 5 5.8 2.6 52.7 17.6 5 8.4 7.2 5 11.4 8.0 UGD4b 6 1.5 17.0 5 3.3 9.3 5 3.7 8.3 5 7.94.4 5 15.2 5.0 UGD1a 4 nd 18.8 4 nd 46.7 4 nd 64.9 4 nd 38.1 5 7.9 44.8UGD gag3349 8 36.6 8 25.4 6 22.9 6 33.1 UGD env 8 9.0 8 2.9 8 13.3 812.5 8 14.3 UGDgag/gp140 5 1.2 18.9 5 2.3 17.6

TABLE 6 Modification of UGD env Gene in Recombinant MVA % non-stainingpassage env gag UGD9 5 0.5 5 0.4 5 0.0 5 0.5

TABLE 7 MVA/UGD4b- Analysis of Non-Staining  gag Plaques #individual plaques  base with mutation gene # sequence MVA/UGD MVA/KEAMVA/TZC p17   28 GGGGG   70 GGGGG n = 1 p24  408 GGGG  530 CCCC n = 1 564 GGGGGG n = 7 n = 16 n = 21  686 GGGG 1050 GGGGGG P7 1133 GGGG p11320 GGGG p6 1361 CCCC 1387 GGGG 1419 GGGG 1473 CCCC Protease 1494 GGGGGRT 1590 GGGGG 1599 GGGGG 2362 GGGG 2380 GGGG 2528 GGGGG 2596 GGGG 2893GGGG 3001 CCCC

TABLE 8 Modification of UGD gagpol Gene in Recombinant MVA %non-staining Passage env gag UGD gag (c.a.) 6 0.9 6 0.0 6 0.5

TABLE 9 Construction of Stable Recombinant MVA Expressing UGD env andgagpol % non-staining Passage env gag UGD4d 11 0.0 0.7

Example 4

This Example demonstrates the use of additional insertion sites forgenerating stable, recombinant MVA viruses. The Del III region of theMVA virus genome contains several non-essential genes, and fragments ofgenes, and thus has historically been used to insert heterologousnucleic acid sequences. Thus, the flanking region around the del IIIinsertion site of MVA was analyzed for the presence of fragmented ornon-essential genes. Genes known to be important for VACV replication insome cells, i.e. A50R DNA ligase and B1R kinase were located about 1 kbpand 1.8 kbp, respectively, from the del III insertion site. We reasonedwe could make this a more stable insertion site if we removed thenon-essential genes flanking the Del III insertion site. To this end, anucleic acid construct (e.g., shuttle vector) with flanking sequencescomprising the 3′ end part of A50R DNA ligase ORF (left), and the 5′ endof the B1R ORE, and promoter (right), was constructed as follows. Thiswould effectively remove the area of non-essential genes between thesetwo important genes when homologous recombination occurred.

A. Preparation of the A50R/B1R Shuttle Vector:

Analysis of the flanking regions around the del III insertion site inthe MVA genome, (bp number 143552, Acambis 3000 Genbank AY603355)revealed that at least two genes known to be important for VACVreplication in some cells. Specifically, A50R DNA ligase (ORF 163;ACAM3000_MVA_(—)163; SEQ ID NO:11) and B1R kinase (ORF167;ACAM3000_MVA_(—)167; SEQ ID NO:16) were located about 1 kbp and 1.8 kbp,respectively, from the del III insertion site. Thus, non-essential orfragmented genes located between ORF 163 and ORF 167 were targeted forremoval. In particular, ORF 164, fragments of A51R-A55, ORF165 (missingthe part of the A56R promoter), ORF 166, and fragmented A57R weretargeted for removal. In order to effect removal these non-essential andfragmented genes, a nucleic acid construct (i.e., a shuttle vector) wasdesigned that would be capable of homologously recombining into the MVAgenome between ORF 163 and ORF 167, thereby removing the interveningsequences. To achieve such recombination, the nucleic acid constructwould comprise one nucleic acid sequence from ACAM3000_MVA_(—)163 (theleft flanking sequence), and one nucleic acid sequence fromACAM3000_MVA_(—)167 (the right flanking sequence). These sequences wouldbe adjacent in the nucleic acid construct, meaning that they would notbe separated by any poxvirus ORF's. More specifically, the left flankwould contain the C terminal end of the A50R ligase ORF and the rightflank would contain the promoter region and the N terminal end of theB1R ORF. The design of the vector is shown in FIG. 26.

To construct the shuttle vector, each flank was created separately. Theleft flank of the restructured Del III vector was constructed first, asfollows.

Plasmid LW-73 (FIG. 7) was digested with EcoRI and XhoI to excise theentire left flank (Flank 1 containing a portion of the I8R gene) alongwith the gene encoding green fluorescent protein (GFP) and directrepeat. The GFP containing fragment was then digested with restrictionenzymes AcsI and SacI to liberate the GFP gene.

To create the left flank containing C-terminal portion of ORF 163, a DNAfragment was amplified from the MVA genome by the polymerase chainreaction (PCR) method using the primers LW470 (SEQ ID NO:23) and LW471(SEQ ID NO:24). PCR amplification was performed using standardconditions. Next, the direct terminal repeat portion of ORF 163 wasamplified from the MVA genome using the primers LW-472 (SEQ ID NO:25)and LW-473 (SEQ ID NO:26). Finally, the vector backbone, with EcoRI andXhoI sites, the GFP gene, with AcsI and SacI sites, the ORF 163 fragment(left flank) containing EcoRI and AscI sites, and the direct repeat fromthe ORF 163 C-terminus region, containing the SacI and XhoI sites, wereligated together to form the interim plasmid #2743.

To create the right flank containing the N-terminal portion of ORF 167,including its promoter region, interim plasmid #2743 was digested withthe restriction enzymes Pst I and HindIII to release the right flank.Next, a DNA fragment was PCR amplified from the MVA genome using theprimers LW-474 (SEQ ID NO:27) and LW-475 (SEQ ID NO:28). This fragmentwas digested with the restriction enzymes Pst I and Hind III, and thedigested fragment ligated into similarly-digested, shuttle vectorbackbone to produce the LW-676 nucleic acid construct. (FIG. 27)

The salient features of pLW-76 are:

1) the vector is designed for insertion of foreign genes between the endof the A50R DNA ligase gene (ORF 163) and the promoter and N terminalportion of the B1R kinase gene (ORF 167) in MVA genome. The left flankconsists of end of A50R ligase gene and right flank consists of promoterand beginning of the B1R kinase.

2) the GFP gene is included for easy initial selection of recombinantvirus.

3) the GFP is flanked by direct repeat of the A50R ligase gene whichallows for transient expression of GFP as the GFP will be lost uponrepeated passage of the recombinant virus. Features 2 and 3 were alsocontained in earlier plasmids used for making MVA/HIV recombinants,pLAS-1 and pLAS-2.

The env gene from Ugandan clade D human immunodeficiency virus (HIV)(isolate AO7412) was then cloned into the new pLW-76 construct. The envcontaining nucleic acid construct was then transfected into cells, andthe cells infected with MVA virus to produce a recombinant MVA virusexpressing the HIV ENV protein rMVA/UGDenv(delIIIrst). This virus wasthen characterized.

When grown in chick embryo fibroblast (CEF) cells, it was observed thatinfection by rMVA/UGDenv(delIIIrst) resulted in syncytial-type,cytoplasmic effect (CPE). This was due to the deletion of thenon-essential A56 hemagglutinin gene during recombination that occurredwithin the restructured del III site. Normal rMVA had a flay focus (FIG.28A), whereas infection with rMVA/UGDenv (delIIrt) resulted in focishowing syncytial formation, progressing to condensed syncytial. (FIG.28B).

rMVA/UGDenv (delIIIrst) was then characterized with regard to thestability of the inserted heterologous nucleic acid sequences. This wasdone by repeatedly passaging the virus in CEF cells, and testing eachgeneration for the presence of expressed HIV ENV protein. Detection ofENV protein was done by screening viral plaques with monoclonalantibodies to the HIV envelope protein. The stability of rMVA/UGDenv(delIIIrst) was compared to a virus containing the env gene in the delII region, and a virus in which the env gene was inserted into thecentral conserved region. The results of this comparison are shown inFIG. 29. The level of ENV protein being expressed was also measured byWestern blot, using monoclonal antibodies to the HIV ENV protein.

FIG. 29 shows that the MVA/UGDenv(del II) was clearly unstable, due todeletions that occurred within the env and extending into the flankingMVA. Viable deletions were prevented when the UGD env was placed betweentwo VACV essential genes, as in MVA/UGDenv(I8/G1). Finally, integrationof the HIV env gene int rMVA/UGDenv(del IIIrst), was observed to bestable at least through 11 passages.

FIG. 30 shows that 11 viral constructs expressed similar amounts of ENVprotein.

Thus, the results of these studies suggest that the del III region ofthe MVA virus genome had been made more stable by restructuring the delIII site by removing the non-essential genes.

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All figures, tables, andappendices, as well as patents, applications, and publications, referredto above, are hereby incorporated by reference.

What is claimed is:
 1. A recombinant modified vaccinia Ankara (MVA)virus comprising a heterologous nucleic acid sequence between twoadjacent essential open reading frames (ORFs) of the MVA virus genome,wherein the MVA virus lacks non-essential ORFs that are present betweenthe corresponding two essential ORFs in the parental MVA virus.
 2. Therecombinant MVA virus of claim 1, wherein the heterologous nucleic acidsequence is in an intergenic region (IGR) located between the twoadjacent, essential ORFs.
 3. The recombinant MVA virus of claim 1,wherein the non-essential ORFs flank the Del III insertion site of theparental MVA virus.
 4. The recombinant MVA virus of claim 1, wherein atleast one of the adjacent essential ORFs has at least 90% identity withSEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:16 or SEQ ID NO:19.
 5. Therecombinant MVA virus of claim 1, wherein one of the adjacent essentialORFs has at least 90% identity with SEQ ID NO:11 or SEQ ID NO:14, andthe other adjacent essential ORF has at least 90% identity with SEQ IDNO:16 or SEQ ID NO:19.
 6. The recombinant MVA virus of claim 1, whereinone of the adjacent essential ORFs comprises SEQ ID NO:11 or SEQ IDNO:14, and the other adjacent essential ORF comprises SEQ ID NO:16 orSEQ ID NO:19.
 7. The recombinant MVA virus of claim 1, wherein one ofthe adjacent essential ORFs is 163 (A50R) and the other adjacentessential ORF is 167 (B1R), using the nomenclature according toCDC/Acambis.
 8. The recombinant MVA virus of claim 1, wherein theheterologous nucleic acid sequence comprises at least one codingsequence under transcriptional control of a transcriptional controlelement.
 9. The recombinant MVA virus of claim 8, wherein the codingsequence encodes one or more proteins.
 10. The recombinant MVA virus ofclaim 8, wherein the transcriptional control element is a poxvirustranscriptional control element.
 11. The recombinant MVA virus of claim8, wherein the coding sequence is derived from human immunodeficiencyvirus (HIV).
 12. The recombinant MVA virus of claim 8, wherein thecoding sequence encodes the HIV envelope protein.
 13. The recombinantMVA virus of claim 8, wherein the coding sequence comprises a nucleotidesequence at least 90% identical to SEQ ID NO: 4, and wherein the encodedprotein is capable of eliciting an immune response to the proteinencoded by SEQ ID NO:4.
 14. The recombinant MVA virus of claim 8,wherein the coding sequence comprises SEQ ID NO:
 4. 15. A method forproducing a stable, recombinant modified vaccinia Ankara (MVA) virus,the method comprising: (a) transfecting a cell with a nucleic acidconstruct comprising; (i) a first nucleic acid sequence comprising an atleast 100 contiguous polynucleotide region that is at least 75%identical to an at least 100 contiguous polynucleotide region from afirst essential MVA virus ORF; and (ii) a second nucleic acid sequencecomprising an at least 100 contiguous polynucleotide region that is atleast 75% identical to an at least 100 contiguous polynucleotide regionfrom a second essential MVA virus ORF; wherein the first and secondessential MVA virus ORFs are separated by at least one non-essential ORFin the MVA virus genome, and wherein the first and second nucleic acidsequences are adjacent to each other in the isolated nucleic acidconstruct; (b) infecting the transfected cell with a MVA virus; (c)culturing the infected cell under conditions suitable to allowhomologous recombination between the nucleic acid construct and the MVAvirus genome; and (d) isolating the recombinant MVA virus.
 16. A methodfor inducing an immune response to a heterologous protein in a subject,comprising administering to the subject the recombinant MVA virus ofclaim 8, wherein the heterologous nucleic acid sequence encodes theheterologous protein.
 17. A method for producing a heterologous proteinin vitro, the method comprising: (a) infecting a host cell with therecombinant MVA virus of claim 8, wherein the heterologous nucleic acidsequence encodes the heterologous protein; (b) cultivating the infectedhost cell under suitable conditions; and (c) isolating the heterologousprotein produced by said host cell.
 18. The recombinant MVA virus ofclaim 1, wherein the two adjacent essential ORFs are selected from thegroup consisting of A50R, B1R, F10L, F12L, F13L, F15L, F17L, E4L, E6L,E8L, E10L, I1L, I3L, I5L, J1R, J3R, D7L, D9L, A24R and A28R.
 19. Therecombinant MVA virus of claim 1, wherein the two adjacent essentialORFs are selected from the group consisting of A50R-B1R, F10L-F12L,F13L-F15L, F15L-F17L, E4L-E6L, E6L-E8L, I3L-I5L, J1R-J3R, D7L-D9L andA24R-A28R.
 20. The recombinant MVA of claim 1, wherein each of theadjacent essential ORFs comprise an amino acid sequence at least 90%identical to the sequence of an essential ORF selected from the groupconsisting of A50R, B1R, F10L, F12L, F13L, F15L, F17L, E4L, E6L, E8L,E10L, I1L, I3L, I5L, J1R, J3R, D7L, D9L, A24R and A28R.